Novel heterodimeric proteins

ABSTRACT

The invention provides novel heterodimeric proteins including heterodimeric antibodies.f5

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/205,248, filed Mar. 11, 2014 which is a continuation of U.S. patentapplication Ser. No. 14/155,334 filed Jan. 14, 2014, which claimspriority to U.S. Provisional Patent Applications No. 61/752,348 filed onJan. 14, 2013, and No. 61/764,954 filed on Feb. 14, 2013, and No.61/780,334 filed on Mar. 13, 2013, and No. 61/818,410 filed on May 1,2013, and No. 61/778,157 filed on Mar. 12, 2013, and No. 61/913,832filed on Dec. 9, 2013, and No. 61/818,153 filed on May 1, 2013 and No.61/913,870 filed on Dec. 9, 2013 the entire contents of which areincorporated herein for all purposes by this reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 8, 2017, isnamed 067461_5161US05_ST25.txt and is 850,139 bytes in size.

INCORPORATION OF RELATED APPLICATIONS

The following applications are incorporated by reference in theirentirety, U.S. Ser. Nos. 61/302,707, 61/732,813, 61/598,686, 61/441,552,61/311,472, 61/450,457, 61/545,498, 61/368,969, 61/391,509, 61/391,515,61/439,263, 61/368,962 and 61/593,846, 61/515,745, 61/752,349,13/194,904, 13/568,028, 13/648,951, 12/875,015, as well as USPublication Nos. 2009/0163,699 and 2006/0024298.

TECHNICAL FIELD

The present invention describes novel immunoglobulin compositions thatsimultaneously co-engage antigens, where both of the antigens are boundmonovalently. The novel immunoglobulins described preferably utilizeheterodimeric Fc regions. Methods of using the novel immunoglobulincompositions, particularly for therapeutic purposes, are also describedherein.

BACKGROUND OF THE INVENTION

Antibody-based therapeutics have been used successfully to treat avariety of diseases, including cancer and autoimmune/inflammatorydisorders. Yet improvements to this class of drugs are still needed,particularly with respect to enhancing their clinical efficacy. Oneavenue being explored is the engineering of additional and novel antigenbinding sites into antibody-based drugs such that a singleimmunoglobulin molecule co-engages two different antigens. Suchnon-native or alternate antibody formats that engage two differentantigens are often referred to as bispecifics. Because the considerablediversity of the antibody variable region (Fv) makes it possible toproduce an Fv that recognizes virtually any molecule, the typicalapproach to bispecific generation is the introduction of new variableregions into the antibody.

A number of alternate antibody formats have been explored for bispecifictargeting (Chames & Baty, 2009, mAbs 1[6]:1-9; Holliger & Hudson, 2005,Nature Biotechnology 23[9]:1126-1136; and Kontermann, 2012 MAbs4(2):182, all of which are expressly incorporated herein by reference).Initially, bispecific antibodies were made by fusing two cell lines thateach produced a single monoclonal antibody (Milstein et al., 1983,Nature 305:537-540). Although the resulting hybrid hybridoma or quadromadid produce bispecific antibodies, they were only a minor population,and extensive purification was required to isolate the desired antibody.An engineering solution to this was the use of antibody fragments tomake bispecifics. Because such fragments lack the complex quaternarystructure of a full length antibody, variable light and heavy chains canbe linked in single genetic constructs. Antibody fragments of manydifferent forms have been generated, including diabodies, single chaindiabodies, tandem scFv's, and Fab₂ bispecifics (Chames & Baty, 2009,mAbs 1[6]:1-9; Holliger & Hudson, 2005, Nature Biotechnology23[9]:1126-1136; expressly incorporated herein by reference). Whilethese formats can be expressed at high levels in bacteria and may havefavorable penetration benefits due to their small size, they clearrapidly in vivo and can present manufacturing obstacles related to theirproduction and stability. A principal cause of these drawbacks is thatantibody fragments typically lack the constant region of the antibodywith its associated functional properties, including larger size, highstability, and binding to various Fc receptors and ligands that maintainlong half-life in serum (i.e., the neonatal Fc receptor FcRn) or serveas binding sites for purification (i.e., protein A and protein G).

More recent work has attempted to address the shortcomings offragment-based bispecifics by engineering dual binding into full lengthantibody-like formats (Wu et al., 2007, Nature Biotechnology25[11]:1290-1297; U.S. Ser. No. 12/477,711; Michaelson et al., 2009,mAbs 1[2]:128-141; PCT/US2008/074693; Zuo et al., 2000, ProteinEngineering 13[5]:361-367; U.S. Ser. No. 09/865,198; Shen et al., 2006,J Biol Chem 281[16]:10706-10714; Lu et al., 2005, J Biol Chem280[20]:19665-19672; PCT/US2005/025472; and Kontermann, 2012 MAbs4(2):182, all of which are expressly incorporated herein by reference).These formats overcome some of the obstacles of the antibody fragmentbispecifics, principally because they contain an Fc region. Onesignificant drawback of these formats is that, because they build newantigen binding sites on top of the homodimeric constant chains, bindingto the new antigen is always bivalent.

For many antigens that are attractive as co-targets in a therapeuticbispecific format, the desired binding is monovalent rather thanbivalent. For many immune receptors, cellular activation is accomplishedby cross-linking of a monovalent binding interaction. The mechanism ofcross-linking is typically mediated by antibody/antigen immunecomplexes, or via effector cell to target cell engagement. For example,the low affinity Fc gamma receptors (FcγRs) such as FcγRIIa, FcγRIIb,and FcγRIIIa bind monovalently to the antibody Fc region. Monovalentbinding does not activate cells expressing these FcγRs; however, uponimmune complexation or cell-to-cell contact, receptors are cross-linkedand clustered on the cell surface, leading to activation. For receptorsresponsible for mediating cellular killing, for example FcγRIIIa onnatural killer (NK) cells, receptor cross-linking and cellularactivation occurs when the effector cell engages the target cell in ahighly avid format (Bowles & Weiner, 2005, J Immunol Methods 304:88-99,expressly incorporated by reference). Similarly, on B cells theinhibitory receptor FcγRIIb downregulates B cell activation only when itengages into an immune complex with the cell surface B-cell receptor(BCR), a mechanism that is mediated by immune complexation of solubleIgG's with the same antigen that is recognized by the BCR (Heyman 2003,Immunol Lett 88[2]:157-161; Smith and Clatworthy, 2010, Nature ReviewsImmunology 10:328-343; expressly incorporated by reference). As anotherexample, CD3 activation of T-cells occurs only when its associatedT-cell receptor (TCR) engages antigen-loaded MHC on antigen presentingcells in a highly avid cell-to-cell synapse (Kuhns et al., 2006,Immunity 24:133-139). Indeed nonspecific bivalent cross-linking of CD3using an anti-CD3 antibody elicits a cytokine storm and toxicity(Perruche et al., 2009, J Immunol 183[2]:953-61; Chatenoud & Bluestone,2007, Nature Reviews Immunology 7:622-632; expressly incorporated byreference). Thus for practical clinical use, the preferred mode of CD3co-engagement for redirected killing of targets cells is monovalentbinding that results in activation only upon engagement with theco-engaged target.

Thus while bispecifics generated from antibody fragments sufferbiophysical and pharmacokinetic hurdles, a drawback of those built withfull length antibody-like formats is that they engage co-target antigensmultivalently in the absence of the primary target antigen, leading tononspecific activation and potentially toxicity. The present inventionsolves this problem by introducing a novel bispecific format thatenables the co-engagement of distinct target antigens.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention provides heterodimericantibodies comprising a first heavy chain comprising a first Fc domainand a single chain Fv region that binds a first antigen. Theheterodimeric antibodies also comprise a second heavy chain comprising asecond Fc domain, a first variable heavy chain and a first variablelight chain, wherein the first and second Fc domains are different, suchthat heterodimerization occurs.

In a further aspect, the pIs of the first heavy chain and the secondheavy chain are at least 0.5 logs apart.

In an additional aspect, the heterodimeric antibodies of the inventionfurther comprise one or two additional antigen binding domains that canbe either or both of an scFv and a Fab.

In a further aspect, the heterodimeric antibodies of the inventioncomprise heterodimerization variants according to the Figures.

In an additional aspect, the heterodimeric antibodies comprise pIvariants in each monomer, with monomer one are ISO(−):

I199T/N203D/K274Q/R355Q/N384S/K392N/V397M/Q419E/DEL44 and the pIsubsitutions in monomer 2 are ISO(+RR): Q196K/I199T/P217R/P228R/N276K.

In additional aspects, the present invention provides heterodimericantibodies wherein one of the heavy chains comprises a variant selectedfrom the group consisting of S119E, K133E, K133Q, R133E (in case ofIgG2-4), R133Q (in case of IgG2-4), T164E, K205E, K205Q, N208D, K210E,K210Q, K274E, K320E, K322E, K326E, K334E, R355E, K392E, Deletion ofK447, adding peptide DEDE at the c-terminus, G137E, N203D, K274Q, R355Q,K392N and Q419E, 349A, 349C, 349E, 349I, 349K, 349S, 349T, 349W, 351E,351K, 354C, 356K, 357K, 364C, 364D, 364E, 364F, 364G, 364H, 364R, 364T,364Y, 366D, 366K, 366S, 366W, 366Y, 368A, 368E, 368K, 368S, 370C, 370D,370E, 370G, 370R, 370S, 370V, 392D, 392E, 394F, 394S, 394W, 394Y, 395T,395V, 396T, 397E, 397S, 397T, 399K, 401K, 405A, 405S, 407T, 407V, 409D,409E, 411D, 411E, 411K, 439D, 349C/364E, 349K/351K, 349K/351K/394F,349K/354C, 349K/394F, 349K/394F/401K, 349K/394Y, 349K/401K, 349K/405A,349T/351E/411E, 349T/394F, 349T/394F/401K, 349T/394F/411E, 349T/405A,349T/411E, 351E/364D, 351E/364D/405A, 351E/364E, 351E/366D,351K/364H/401K, 351K/366K, 364D/370G, 364D/394F, 364E/405A, 364E/405S,364E/411E, 364E/411E/405A, 364H/394F, 364H/401K, 364H/401K/405A,364H/405A, 364H/405A/411E, 364Y/370R, 370E/411E, 370R/411K,395T/397S/405A and 397S/405A.

In a further aspect, the invention provides compositions comprising ananti-CD3 variable region having a sequence comprising a vhCDR1 havingSEQ ID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ IDNO:416, a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 anda vlCDR3 having SEQ ID NO:430.

In an additional aspect, the invention provides compositions comprisingan anti-CD3 variable region having a sequence comprising a vhCDR1 havingthe sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), avhCDR2 having the sequenceR-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Yor A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436),a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, whereinXaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 havingthe sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G,R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ IDNO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, whereinXaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and avlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A orL and Xaa2 is L or H (SEQ ID NO:440).

In a further aspect, the anti-CD3 variable region has a sequenceselected from the group consisting of: a) a sequence comprising a vhCDR1having SEQ ID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQID NO:416, a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425and a vlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 havingSEQ ID NO:412, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ IDNO:416, a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 anda vlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:414, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:417,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:418,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:421, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:422, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:427 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:428 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:431; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:423, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:432; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:424, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:432; a sequence comprising a vhCDR1 having SEQID NO:412, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:417,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:412, a vhCDR2 having SEQ ID NO:414, a vhCDR3 having SEQ ID NO:419,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:415, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:415, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:417,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:419,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:430; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:417,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:433; a sequence comprising a vhCDR1 having SEQID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ ID NO:416,a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 and avlCDR3 having SEQ ID NO:433 and a sequence comprising a vhCDR1 havingSEQ ID NO:411, a vhCDR2 having SEQ ID NO:434, a vhCDR3 having SEQ IDNO:416, a vlCDR1 having SEQ ID NO:420, a vlCDR2 having SEQ ID NO:425 anda vlCDR3 having SEQ ID NO:430.

One preferred aspect provides an anti-CD3 variable region having asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:417, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:433.

In an additional aspect, the invention provides amino acid compositionswherein the composition comprises a first amino acid sequence comprisingthe variable heavy CDRs and a second amino acid sequence comprising thevariable light CDRs. In this aspect, the anti-CD3 variable regioncomprises a variable heavy region and a variable light region selectedfrom the group consisting of: SEQ ID NOs: 5 and 6; SEQ ID NOs: 9 and 10;SEQ ID NOs: 13 and 14; SEQ ID NOs: 17 and 18; SEQ ID NOs: 21 and 22; SEQID NOs: 25 and 26; SEQ ID NOs: 29 and 30; SEQ ID NOs: 33 and 34; SEQ IDNOs: 37 and 38; SEQ ID NOs: 41 and 42; SEQ ID NOs: 45 and 46; SEQ IDNOs: 49 and 50; SEQ ID NOs: 53 and 54; SEQ ID NOs: 57 and 58; SEQ IDNOs: 61 and 62; SEQ ID NOs: 65 and 66; SEQ ID NOs: 69 and 70; SEQ IDNOs: 73 and 74; SEQ ID NOs: 77 and 78; SEQ ID NOs: 81 and 82; SEQ IDNOs: 85 and 86; SEQ ID NOs: 89 and 90; SEQ ID NOs: 93 and 94; SEQ IDNOs: 97 and 98; SEQ ID NOs: 101 and 102; SEQ ID NOs: 105 and 106; SEQ IDNOs: 109 and 110; SEQ ID NOs: 113 and 114; SEQ ID NOs: 117 and 118; SEQID NOs: 121 and 122; SEQ ID NOs: 125 and 126; SEQ ID NOs: 129 and 130;SEQ ID NOs: 133 and 134; SEQ ID NOs: 137 and 138; SEQ ID NOs: 141 and142; SEQ ID NOs: 145 and 146; SEQ ID NOs: 149 and 150; SEQ ID NOs: 153and 154; SEQ ID NOs: 157 and 158; SEQ ID NOs: 161 and 162; SEQ ID NOs:165 and 166; SEQ ID NOs: 169 and 170; SEQ ID NOs: 173 and 174; SEQ IDNOs: 177 and 178; SEQ ID NOs: 181 and 182; SEQ ID NOs: 185 and 186; SEQID NOs: 189 and 190; SEQ ID NOs: 193 and 194; SEQ ID NOs: 197 and 198;SEQ ID NOs: 201 and 202; SEQ ID NOs: 205 and 206; SEQ ID NOs: 209 and210; SEQ ID NOs: 213 and 214; SEQ ID NOs: 217 and 218; SEQ ID NOs: 221and 222; SEQ ID NOs: 225 and 226; SEQ ID NOs: 229 and 230; SEQ ID NOs:233 and 234; SEQ ID NOs: 237 and 238; SEQ ID NOs: 241 and 242; SEQ IDNOs: 245 and 246; SEQ ID NOs: 249 and 250; SEQ ID NOs: 253 and 254; SEQID NOs: 257 and 258; SEQ ID NOs: 261 and 262; SEQ ID NOs: 265 and 266;SEQ ID NOs: 269 and 270; SEQ ID NOs: 273 and 274; SEQ ID NOs: 277 and278; SEQ ID NOs: 281 and 282; SEQ ID NOs: 285 and 286; SEQ ID NOs: 289and 290; SEQ ID NOs: 293 and 294; SEQ ID NOs: 297 and 298; SEQ ID NOs:301 and 302; SEQ ID NOs: 305 and 306; SEQ ID NOs: 309 and 310; SEQ IDNOs: 313 and 314; SEQ ID NOs: 317 and 318; SEQ ID NOs: 321 and 322; SEQID NOs: 325 and 326; SEQ ID NOs: 329 and 330; SEQ ID NOs: 333 and 334;SEQ ID NOs: 337 and 338; SEQ ID NOs: 341 and 342; SEQ ID NOs: 345 and346; SEQ ID NOs: 349 and 350; SEQ ID NOs: 353 and 354; SEQ ID NOs: 357and 358; SEQ ID NOs: 361 and 362; SEQ ID NOs: 365 and 366; SEQ ID NOs:369 and 370; SEQ ID NOs: 373 and 374; SEQ ID NOs: 377 and 378; SEQ IDNOs: 381 and 382; SEQ ID NOs: 385 and 386; SEQ ID NOs: 389 and 390; SEQID NOs: 393 and 394; SEQ ID NOs: 397 and 398; SEQ ID NOs: 401 and 402;SEQ ID NOs: 405 and 406; SEQ ID NOs: 409 and 410.

In some aspects, the invention provides compositions including scFv, andscFv charged linkers. In one embodiment, the charged scFv linker has apositive charge from 3 to 8 and is selected from the group consisting ofSEQ ID NO:s 443 to 451. In another embodiment, the charged scFv linkerhas a negative charge from 3 to 8 and is selected from the groupconsisting of SEQ ID NO:s 453 to 459.

In a further aspect, the anti-CD3 scFv has a sequence selected from thegroup consisting of: SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ IDNO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQID NO: 36; SEQ ID NO: 40; SEQ ID NO: 44; SEQ ID NO: 48; SEQ ID NO: 52;SEQ ID NO: 56; SEQ ID NO: 60; SEQ ID NO: 64; SEQ ID NO: 68; SEQ ID NO:72; SEQ ID NO: 76; SEQ ID NO: 80; SEQ ID NO: 84; SEQ ID NO: 88; SEQ IDNO: 92; SEQ ID NO: 96; SEQ ID NO: 100; SEQ ID NO: 104; SEQ ID NO: 108;SEQ ID NO: 112; SEQ ID NO: 116; SEQ ID NO: 120; SEQ ID NO: 124; SEQ IDNO: 128; SEQ ID NO: 132; SEQ ID NO: 136; SEQ ID NO: 140; SEQ ID NO: 144;SEQ ID NO: 148; SEQ ID NO: 152; SEQ ID NO: 156; SEQ ID NO: 160; SEQ IDNO: 164; SEQ ID NO: 168; SEQ ID NO: 172; SEQ ID NO: 176; SEQ ID NO: 180;SEQ ID NO: 184; SEQ ID NO: 188; SEQ ID NO: 192; SEQ ID NO: 196; SEQ IDNO: 200; SEQ ID NO: 204; SEQ ID NO: 208; SEQ ID NO: 212; SEQ ID NO: 216;SEQ ID NO: 220; SEQ ID NO: 224; SEQ ID NO: 228; SEQ ID NO: 232; SEQ IDNO: 236; SEQ ID NO: 240; SEQ ID NO: 244; SEQ ID NO: 248; SEQ ID NO: 252;SEQ ID NO: 256; SEQ ID NO: 260; SEQ ID NO: 264; SEQ ID NO: 268; SEQ IDNO: 272; SEQ ID NO: 276; SEQ ID NO: 280; SEQ ID NO: 284; SEQ ID NO: 288;SEQ ID NO: 292; SEQ ID NO: 296; SEQ ID NO: 300; SEQ ID NO: 304; SEQ IDNO: 308; SEQ ID NO: 312; SEQ ID NO: 316; SEQ ID NO: 320; SEQ ID NO: 324;SEQ ID NO: 328; SEQ ID NO: 332; SEQ ID NO: 336; SEQ ID NO: 340; SEQ IDNO: 344; SEQ ID NO: 348; SEQ ID NO: 352; SEQ ID NO: 356; SEQ ID NO: 360;SEQ ID NO: 364; SEQ ID NO: 368; SEQ ID NO: 372; SEQ ID NO: 376; SEQ IDNO: 380; SEQ ID NO: 384; SEQ ID NO: 388; SEQ ID NO: 392; SEQ ID NO: 396;SEQ ID NO: 400; SEQ ID NO: 404; SEQ ID NO: 408. In some embodiments, thescFv linkers of these sequences can be exchanged for a charged scFv.

In an additional aspect, the invention provides a nucleic acidcomposition encoding an anti-CD3 variable region comprising a variableheavy region and a variable light region selected from the groupconsisting of SEQ ID NOs: 5 and 6; SEQ ID NOs: 9 and 10; SEQ ID NOs: 13and 14; SEQ ID NOs: 17 and 18; SEQ ID NOs: 21 and 22; SEQ ID NOs: 25 and26; SEQ ID NOs: 29 and 30; SEQ ID NOs: 33 and 34; SEQ ID NOs: 37 and 38;SEQ ID NOs: 41 and 42; SEQ ID NOs: 45 and 46; SEQ ID NOs: 49 and 50; SEQID NOs: 53 and 54; SEQ ID NOs: 57 and 58; SEQ ID NOs: 61 and 62; SEQ IDNOs: 65 and 66; SEQ ID NOs: 69 and 70; SEQ ID NOs: 73 and 74; SEQ IDNOs: 77 and 78; SEQ ID NOs: 81 and 82; SEQ ID NOs: 85 and 86; SEQ IDNOs: 89 and 90; SEQ ID NOs: 93 and 94; SEQ ID NOs: 97 and 98; SEQ IDNOs: 101 and 102; SEQ ID NOs: 105 and 106; SEQ ID NOs: 109 and 110; SEQID NOs: 113 and 114; SEQ ID NOs: 117 and 118; SEQ ID NOs: 121 and 122;SEQ ID NOs: 125 and 126; SEQ ID NOs: 129 and 130; SEQ ID NOs: 133 and134; SEQ ID NOs: 137 and 138; SEQ ID NOs: 141 and 142; SEQ ID NOs: 145and 146; SEQ ID NOs: 149 and 150; SEQ ID NOs: 153 and 154; SEQ ID NOs:157 and 158; SEQ ID NOs: 161 and 162; SEQ ID NOs: 165 and 166; SEQ IDNOs: 169 and 170; SEQ ID NOs: 173 and 174; SEQ ID NOs: 177 and 178; SEQID NOs: 181 and 182; SEQ ID NOs: 185 and 186; SEQ ID NOs: 189 and 190;SEQ ID NOs: 193 and 194; SEQ ID NOs: 197 and 198; SEQ ID NOs: 201 and202; SEQ ID NOs: 205 and 206; SEQ ID NOs: 209 and 210; SEQ ID NOs: 213and 214; SEQ ID NOs: 217 and 218; SEQ ID NOs: 221 and 222; SEQ ID NOs:225 and 226; SEQ ID NOs: 229 and 230; SEQ ID NOs: 233 and 234; SEQ IDNOs: 237 and 238; SEQ ID NOs: 241 and 242; SEQ ID NOs: 245 and 246; SEQID NOs: 249 and 250; SEQ ID NOs: 253 and 254; SEQ ID NOs: 257 and 258;SEQ ID NOs: 261 and 262; SEQ ID NOs: 265 and 266; SEQ ID NOs: 269 and270; SEQ ID NOs: 273 and 274; SEQ ID NOs: 277 and 278; SEQ ID NOs: 281and 282; SEQ ID NOs: 285 and 286; SEQ ID NOs: 289 and 290; SEQ ID NOs:293 and 294; SEQ ID NOs: 297 and 298; SEQ ID NOs: 301 and 302; SEQ IDNOs: 305 and 306; SEQ ID NOs: 309 and 310; SEQ ID NOs: 313 and 314; SEQID NOs: 317 and 318; SEQ ID NOs: 321 and 322; SEQ ID NOs: 325 and 326;SEQ ID NOs: 329 and 330; SEQ ID NOs: 333 and 334; SEQ ID NOs: 337 and338; SEQ ID NOs: 341 and 342; SEQ ID NOs: 345 and 346; SEQ ID NOs: 349and 350; SEQ ID NOs: 353 and 354; SEQ ID NOs: 357 and 358; SEQ ID NOs:361 and 362; SEQ ID NOs: 365 and 366; SEQ ID NOs: 369 and 370; SEQ IDNOs: 373 and 374; SEQ ID NOs: 377 and 378; SEQ ID NOs: 381 and 382; SEQID NOs: 385 and 386; SEQ ID NOs: 389 and 390; SEQ ID NOs: 393 and 394;SEQ ID NOs: 397 and 398; SEQ ID NOs: 401 and 402; SEQ ID NOs: 405 and406; SEQ ID NOs: 409 and 410.

In some aspects, the nucleic acid composition comprises a first nucleicacid encoding a variant heavy region and a second nucleic acid encodinga variable light region.

In additional aspects, the invention provides nucleic acid compositionsencoding scFv amino acid sequence selected from the group consisting ofSEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20;SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 32; SEQ ID NO: 36; SEQ ID NO:40; SEQ ID NO: 44; SEQ ID NO: 48; SEQ ID NO: 52; SEQ ID NO: 56; SEQ IDNO: 60; SEQ ID NO: 64; SEQ ID NO: 68; SEQ ID NO: 72; SEQ ID NO: 76; SEQID NO: 80; SEQ ID NO: 84; SEQ ID NO: 88; SEQ ID NO: 92; SEQ ID NO: 96;SEQ ID NO: 100; SEQ ID NO: 104; SEQ ID NO: 108; SEQ ID NO: 112; SEQ IDNO: 116; SEQ ID NO: 120; SEQ ID NO: 124; SEQ ID NO: 128; SEQ ID NO: 132;SEQ ID NO: 136; SEQ ID NO: 140; SEQ ID NO: 144; SEQ ID NO: 148; SEQ IDNO: 152; SEQ ID NO: 156; SEQ ID NO: 160; SEQ ID NO: 164; SEQ ID NO: 168;SEQ ID NO: 172; SEQ ID NO: 176; SEQ ID NO: 180; SEQ ID NO: 184; SEQ IDNO: 188; SEQ ID NO: 192; SEQ ID NO: 196; SEQ ID NO: 200; SEQ ID NO: 204;SEQ ID NO: 208; SEQ ID NO: 212; SEQ ID NO: 216; SEQ ID NO: 220; SEQ IDNO: 224; SEQ ID NO: 228; SEQ ID NO: 232; SEQ ID NO: 236; SEQ ID NO: 240;SEQ ID NO: 244; SEQ ID NO: 248; SEQ ID NO: 252; SEQ ID NO: 256; SEQ IDNO: 260; SEQ ID NO: 264; SEQ ID NO: 268; SEQ ID NO: 272; SEQ ID NO: 276;SEQ ID NO: 280; SEQ ID NO: 284; SEQ ID NO: 288; SEQ ID NO: 292; SEQ IDNO: 296; SEQ ID NO: 300; SEQ ID NO: 304; SEQ ID NO: 308; SEQ ID NO: 312;SEQ ID NO: 316; SEQ ID NO: 320; SEQ ID NO: 324; SEQ ID NO: 328; SEQ IDNO: 332; SEQ ID NO: 336; SEQ ID NO: 340; SEQ ID NO: 344; SEQ ID NO: 348;SEQ ID NO: 352; SEQ ID NO: 356; SEQ ID NO: 360; SEQ ID NO: 364; SEQ IDNO: 368; SEQ ID NO: 372; SEQ ID NO: 376; SEQ ID NO: 380; SEQ ID NO: 384;SEQ ID NO: 388; SEQ ID NO: 392; SEQ ID NO: 396; SEQ ID NO: 400; SEQ IDNO: 404; SEQ ID NO: 408. In some embodiments, the scFv linkers of thesesequences can be exchanged for a charged scFv.

In an additional aspect, the invention provides host cells comprisingthe expression vectors and nucleic acids encoding the compositions ofthe invention.

In a further aspect, the anti-CD3 variable region is not SEQ ID NO:4.

In an additional aspect, the invention provides heterodimeric antibodiescomprising: a first heavy chain comprising i) a first Fc domain and asingle chain Fv region (scFv) that binds CD3 comprising a vhCDR1 havingthe sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), avhCDR2 having the sequenceR-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Yor A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436),a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, whereinXaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 havingthe sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G,R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ IDNO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, whereinXaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and avlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A orL and Xaa2 is L or H (SEQ ID NO:440). The heterodimeric antibody alsocomprises a second heavy chain comprising a second Fc domain, a firstvariable heavy chain and a first variable light chain, wherein saidfirst and second Fc domains are different. In this aspect, the anti-CD3variable region can have a sequence selected from the group consistingof: a sequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 havingSEQ ID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ IDNO:420, a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430;a sequence comprising a vhCDR1 having SEQ ID NO:412, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:414, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:417, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:418, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:421,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:422,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:427 and a vlCDR3 having SEQ ID NO:430;sequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:428 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:431; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:423,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:432; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:424,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:432; asequence comprising a vhCDR1 having SEQ ID NO:412, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:417, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:412, a vhCDR2 having SEQID NO:414, a vhCDR3 having SEQ ID NO:419, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:415, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:415, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:417, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:419, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:417, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:433; asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:413, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:433 and asequence comprising a vhCDR1 having SEQ ID NO:411, a vhCDR2 having SEQID NO:434, a vhCDR3 having SEQ ID NO:416, a vlCDR1 having SEQ ID NO:420,a vlCDR2 having SEQ ID NO:425 and a vlCDR3 having SEQ ID NO:430.

In some aspects, the composition comprises a first amino acid sequencecomprising the variable heavy CDRs and a second amino acid sequencecomprising the variable light CDRs.

In one aspect of the Triple F format, the invention provides Triple Fheterodimeric antibodies comprising an anti-CD3 variable regioncomprises a variable heavy region and a variable light region selectedfrom the group consisting of: SEQ ID NOs: 5 and 6; SEQ ID NOs: 9 and 10;SEQ ID NOs: 13 and 14; SEQ ID NOs: 17 and 18; SEQ ID NOs: 21 and 22; SEQID NOs: 25 and 26; SEQ ID NOs: 29 and 30; SEQ ID NOs: 33 and 34; SEQ IDNOs: 37 and 38; SEQ ID NOs: 41 and 42; SEQ ID NOs: 45 and 46; SEQ IDNOs: 49 and 50; SEQ ID NOs: 53 and 54; SEQ ID NOs: 57 and 58; SEQ IDNOs: 61 and 62; SEQ ID NOs: 65 and 66; SEQ ID NOs: 69 and 70; SEQ IDNOs: 73 and 74; SEQ ID NOs: 77 and 78; SEQ ID NOs: 81 and 82; SEQ IDNOs: 85 and 86; SEQ ID NOs: 89 and 90; SEQ ID NOs: 93 and 94; SEQ IDNOs: 97 and 98; SEQ ID NOs: 101 and 102; SEQ ID NOs: 105 and 106; SEQ IDNOs: 109 and 110; SEQ ID NOs: 113 and 114; SEQ ID NOs: 117 and 118; SEQID NOs: 121 and 122; SEQ ID NOs: 125 and 126; SEQ ID NOs: 129 and 130;SEQ ID NOs: 133 and 134; SEQ ID NOs: 137 and 138; SEQ ID NOs: 141 and142; SEQ ID NOs: 145 and 146; SEQ ID NOs: 149 and 150; SEQ ID NOs: 153and 154; SEQ ID NOs: 157 and 158; SEQ ID NOs: 161 and 162; SEQ ID NOs:165 and 166; SEQ ID NOs: 169 and 170; SEQ ID NOs: 173 and 174; SEQ IDNOs: 177 and 178; SEQ ID NOs: 181 and 182; SEQ ID NOs: 185 and 186; SEQID NOs: 189 and 190; SEQ ID NOs: 193 and 194; SEQ ID NOs: 197 and 198;SEQ ID NOs: 201 and 202; SEQ ID NOs: 205 and 206; SEQ ID NOs: 209 and210; SEQ ID NOs: 213 and 214; SEQ ID NOs: 217 and 218; SEQ ID NOs: 221and 222; SEQ ID NOs: 225 and 226; SEQ ID NOs: 229 and 230; SEQ ID NOs:233 and 234; SEQ ID NOs: 237 and 238; SEQ ID NOs: 241 and 242; SEQ IDNOs: 245 and 246; SEQ ID NOs: 249 and 250; SEQ ID NOs: 253 and 254; SEQID NOs: 257 and 258; SEQ ID NOs: 261 and 262; SEQ ID NOs: 265 and 266;SEQ ID NOs: 269 and 270; SEQ ID NOs: 273 and 274; SEQ ID NOs: 277 and278; SEQ ID NOs: 281 and 282; SEQ ID NOs: 285 and 286; SEQ ID NOs: 289and 290; SEQ ID NOs: 293 and 294; SEQ ID NOs: 297 and 298; SEQ ID NOs:301 and 302; SEQ ID NOs: 305 and 306; SEQ ID NOs: 309 and 310; SEQ IDNOs: 313 and 314; SEQ ID NOs: 317 and 318; SEQ ID NOs: 321 and 322; SEQID NOs: 325 and 326; SEQ ID NOs: 329 and 330; SEQ ID NOs: 333 and 334;SEQ ID NOs: 337 and 338; SEQ ID NOs: 341 and 342; SEQ ID NOs: 345 and346; SEQ ID NOs: 349 and 350; SEQ ID NOs: 353 and 354; SEQ ID NOs: 357and 358; SEQ ID NOs: 361 and 362; SEQ ID NOs: 365 and 366; SEQ ID NOs:369 and 370; SEQ ID NOs: 373 and 374; SEQ ID NOs: 377 and 378; SEQ IDNOs: 381 and 382; SEQ ID NOs: 385 and 386; SEQ ID NOs: 389 and 390; SEQID NOs: 393 and 394; SEQ ID NOs: 397 and 398; SEQ ID NOs: 401 and 402;SEQ ID NOs: 405 and 406; SEQ ID NOs: 409 and 410.

In one aspect of the Triple F format, the invention provides Triple Fheterodimeric antibodies comprising an anti-CD3 scFv having a sequenceselected from the group consisting of: SEQ ID NO: 4; SEQ ID NO: 8; SEQID NO: 12; SEQ ID NO: 16; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28;SEQ ID NO: 32; SEQ ID NO: 36; SEQ ID NO: 40; SEQ ID NO: 44; SEQ ID NO:48; SEQ ID NO: 52; SEQ ID NO: 56; SEQ ID NO: 60; SEQ ID NO: 64; SEQ IDNO: 68; SEQ ID NO: 72; SEQ ID NO: 76; SEQ ID NO: 80; SEQ ID NO: 84; SEQID NO: 88; SEQ ID NO: 92; SEQ ID NO: 96; SEQ ID NO: 100; SEQ ID NO: 104;SEQ ID NO: 108; SEQ ID NO: 112; SEQ ID NO: 116; SEQ ID NO: 120; SEQ IDNO: 124; SEQ ID NO: 128; SEQ ID NO: 132; SEQ ID NO: 136; SEQ ID NO: 140;SEQ ID NO: 144; SEQ ID NO: 148; SEQ ID NO: 152; SEQ ID NO: 156; SEQ IDNO: 160; SEQ ID NO: 164; SEQ ID NO: 168; SEQ ID NO: 172; SEQ ID NO: 176;SEQ ID NO: 180; SEQ ID NO: 184; SEQ ID NO: 188; SEQ ID NO: 192; SEQ IDNO: 196; SEQ ID NO: 200; SEQ ID NO: 204; SEQ ID NO: 208; SEQ ID NO: 212;SEQ ID NO: 216; SEQ ID NO: 220; SEQ ID NO: 224; SEQ ID NO: 228; SEQ IDNO: 232; SEQ ID NO: 236; SEQ ID NO: 240; SEQ ID NO: 244; SEQ ID NO: 248;SEQ ID NO: 252; SEQ ID NO: 256; SEQ ID NO: 260; SEQ ID NO: 264; SEQ IDNO: 268; SEQ ID NO: 272; SEQ ID NO: 276; SEQ ID NO: 280; SEQ ID NO: 284;SEQ ID NO: 288; SEQ ID NO: 292; SEQ ID NO: 296; SEQ ID NO: 300; SEQ IDNO: 304; SEQ ID NO: 308; SEQ ID NO: 312; SEQ ID NO: 316; SEQ ID NO: 320;SEQ ID NO: 324; SEQ ID NO: 328; SEQ ID NO: 332; SEQ ID NO: 336; SEQ IDNO: 340; SEQ ID NO: 344; SEQ ID NO: 348; SEQ ID NO: 352; SEQ ID NO: 356;SEQ ID NO: 360; SEQ ID NO: 364; SEQ ID NO: 368; SEQ ID NO: 372; SEQ IDNO: 376; SEQ ID NO: 380; SEQ ID NO: 384; SEQ ID NO: 388; SEQ ID NO: 392;SEQ ID NO: 396; SEQ ID NO: 400; SEQ ID NO: 404; SEQ ID NO: 408. In thisaspect, the scFv linker can be standard or a charged scFv linker asdescribed herein.

In a further aspect, the invention provides Triple F formatheterodimeric antibodies comprising a first heavy chain comprising i) afirst Fc domain and a single chain Fv region (scFv) that binds a firstantigen, wherein the scFv comprises a charged scFv linker; and a secondheavy chain comprising: a second Fc domain; a first variable heavychain; and a first variable light chain. The charged scFv linker canhave a positive charge from 3 to 8 and is selected from the groupconsisting of SEQ ID NO: s 443 to 451 or a negative charge from 3 to 8and is selected from the group consisting of SEQ ID NO:s 453 to 459.

In an additional embodiment, Triple F format heterodimeric antibodieswith charged scFv linkers can also include first and second Fc domainseach comprising at least one heterodimerization variant as describedherein.

In a further aspect, the Triple F format heterodimeric antibodiescomprises a first heavy chain comprising i) a first Fc domain, and ii) asingle chain Fv region (scFv) that binds a first antigen; and a secondheavy chain comprising i) a second Fc domain, a first variable heavychain; and a first variable light chain; wherein said first and secondFc domains comprise a variant set selected from the variants listed inFIG. 9. These include sets selected from the group consisting of:L368D/K370S and S364K; L368D/K370S and S364K/E357L; L368D/K370S andS364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370Sand S364K/E357Q; and K370S and S364K/E357Q. Additionalheterodimerization variants can be independently and optionally includedand selected from variants outlined in any of FIG. 9A, 9B, 9C or 33.These compositions can further comprise ablation variants, pI variants,charged variants, isotypic variants, etc.

In a further aspect, the invention provides Triple F formatheterodimeric antibodies comprising: a) a first heavy chain comprisingi) a first Fc domain and ii) a single chain Fv region (scFv) that bindsa first antigen; and a second heavy chain comprising: i) a second Fcdomain; ii) a first variable heavy chain; and iii) a first variablelight chain; wherein one of the Fc domains comprises one or more Fcγreceptor ablation variants. These ablation variants are depicted in FIG.35, and each can be independently and optionally included or excluded,with preferred aspects utilizing ablation variants selected from thegroup consisting of G236R/L328R, E233P/L234V/L235A/G236del/S239K,E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del.Additional heterodimerization variants can be independently andoptionally added.

In an additional aspect, the invention provides Triple F formatheterodimeric antibodies comprising: a first heavy chain comprising: i)a first Fc domain; and ii) a single chain Fv region that binds a firstantigen; and a second heavy chain comprising: i) a second Fc domain; ii)a first variable heavy chain; and iii) a first variable light chain;wherein the first and second Fc domains comprise a set of variants asshown in Figures, 9, 32 or 33.

In a further aspect, the invention provides dual scFv heterodimericantibodies comprising a first heavy chain comprising: i) a first Fcdomain; and ii) a first single chain Fv region (scFv) that binds a firstantigen; wherein the first scFv comprises a first charged scFv linker;and a second heavy chain comprising: i) a second Fc domain; ii) a secondscFv that binds a second antigen. IN this aspect, the first charged scFvlinker has a positive charge from 3 to 8 and is selected from the groupconsisting of SEQ ID NO:s 443 to 451 or a negative charge from 3 to 8and is selected from the group consisting of SEQ ID NO:s 453 to 459.

In an additional aspect, both scFv linkers are charged and they haveopposite charges.

In a further aspect, the invention provides methods of making aheterodimeric antibody comprising: a) providing a first nucleic acidencoding a first heavy chain comprising: i) a first heavy chaincomprising: a first Fc domain; and a single chain Fv region (scFv) thatbinds a first antigen; wherein said scFv comprises a charged linker; andproviding a second nucleic acid encoding a second heavy chaincomprising: a second Fc domain; a first variable heavy chain; andproviding a third nucleic acid comprising a light chain; expressing saidfirst, second and third nucleic acids in a host cell to produce a first,second and third amino acid sequence, respectively; loading said first,second and third amino acid sequences onto an ion exchange column; andcollecting the heterodimeric fraction.

The present invention describes methods for generating the novelcompositions of the invention. The present invention describespurification methods for the immunogloublins herein, particularlymethods for separating heterodimeric and homodimeric protein species.Also described are methods of testing the immunoglobulins herein,including in vitro and in vitro experiments.

The present invention provides isolated nucleic acids encoding the novelimmunoglobulin compositions described herein. The present inventionprovides vectors comprising said nucleic acids, optionally, operablylinked to control sequences. The present invention provides host cellscontaining the vectors, and methods for producing and optionallyrecovering the immunoglobulin compositions.

The present invention provides compositions comprising immunoglobulinpolypeptides described herein, and a physiologically or pharmaceuticallyacceptable carrier or diluent.

The present invention contemplates therapeutic and diagnostic uses forthe immunoglobulin polypeptides disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict stability-optimized, humanized anti-CD3 variantscFvs. Substitutions are given relative to the H1_L1.4 scFv sequence.Amino acid numbering is Kabat numbering.

FIGS. 2A-2YY depict the amino acid sequences of stability-optimized,humanized anti-CD3 variant scFvs, variable heavy and variable lightsequences. (Note also that the first sequence is the histidine taggedversion for ease of purification). CDRs are underlined. It should beunderstood that the increased stability of the optimized variable andoptimized light chains (as well as the scFv chains) can be attributed toframework regions as well as the CDRs. Thus, it should be understoodthat the disclosure of the entire variable region includes thedisclosure of the framework regions, although they are not separatelynumbered. In addition, the scFv linkers are shown in grey. Each scFvlinker can be replaced with a charged scFv linker as depicted in FIG. 7.That is, any charged scFv linker, whether positive or negative,including those depicted in FIG. 7, can be substituted for thehighlighted region in FIGS. 2A to 2YY.

FIGS. 3A-3B show yields after Ni-NTA purification and meltingtemperatures (Tm) as determined by DSF (Differential ScanningFluorimetry) of stability-optimized, humanized anti-CD3 variant scFvs.Similar DSF stability measurements for the I2C anti-CD3 variable regionin a comparable scFv format gave a Tm of 59° C.

FIGS. 4A-4B show fold improvement (relative to H1_L1.4 scFv) in cellsurface binding affinities (IC50) of stability-optimized, humanizedanti-CD3 variant scFvs as determined in a competitive binding experimentusing purified human T cells from PBMCs.

FIG. 5. Figure depicts the sequence of human CD3.

FIGS. 6A-6I depict a collation of all the CD3 vhCDR1-3 and vlCDR1-3sequences useful in the present invention.

FIG. 7 depicts suitable positive and negatively charged scFv linkers. Asingle prior art scFv linker with a single charge is referenced as“Whitlow”, from Whitlow et al., Protein Engineering 6(8):989-995 (1993).It should be noted that this linker was used for reducing aggregationand enhancing proteolytic stability in scFvs.

FIG. 8 depicts the sequences of a preferred embodiment of the invention.The variable regions are underlined, and the charged scFv linker is ingrey.

FIGS. 9A-9D depict novel steric variants. As will be understood by thosein the art, the first column of each table represents “corresponding”monomer pairs; that is, monomer 1 has 405A and the corresponding stericvariant is 394F. It is important to note that in the context of theasymmetrical triple F format, either monomer can have either variant.That is, the scFv monomer can be monomer 1 or monomer 2. Again, thesesets can be optionally and independently combined with other stericvariants as well as other heterodimerization variants including chargepairs, isotypic variants, isosteric variants, pI variants, etc, as longas some “strandedness” is maintained. In addition, the “monomer” refersto the Fc domains; that is, in the triple F format, one monomer is thescFv construct and the other monomer is the Fab construct, despite thefact that there are actually two amino acid sequences that comprise theFab construct (the heavy and light chains). show a number of suitablesteric or “skew” variants of use in the present invention. FIG. 9depicts a number of steric variants that can be used alone or incombination with pI variants (as is true of all the variants in FIG. 9);however, as will be appreciated by those in the art, if there are pIvariants, the “strandedness” of the pI variants and steric variantsshould be maintained. That is, if for example the pI variantsS364K/E357Q (monomer 1) and L368D/K370S (monomer 2) are to be combinedwith FIG. 29C variants, the pI of the steric variants should beconsidered and assigned to the correct monomer. That is, steric variantsthat alter charge (T411E) for example, are added to the “negative”monomer.

FIG. 10 depicts a list of engineered heterodimer-skewing (e.g. “stericheterodimerization”) Fc variants with heterodimer yields (determined byHPLC-CIEX) and thermal stabilities (determined by DSC). Not determinedthermal stability is denoted by “n.d.”.

FIG. 11A-11C. Illustration of the “triple F” format for bispecificimmunoglobulins. FIG. 11A shows a scFv-Fc format. FIG. 11C depicts amore standard bispecific format, also utilizing the pI variants of theinvention (and optionally and independently the other heterodimerizationvariants). FIG. 11B shows the “triple F” format (sometimes also referredto as the “bottle-opener” configuration; (and optionally andindependently the other heterodimerization variants).

FIGS. 12A-12E depict a variety of “higher multispecificity” embodimentsof the present invention. FIG. 12A shows a “triple F” configuration withanother scFv attached to the Fab monomer (this one, along with FIG. 2D,has a greater molecular weight differential as well). FIG. 12B depicts a“triple F” with another scFv attached to the scFv monomer. FIG. 12Cdepicts a “three scFv” format. FIG. 12D depicts an additional Fabattached to the Fab monomer. FIG. 12E depicts a Fab hooked to one of thescFv monomers.

FIGS. 13A-13E show additional varieties of “higher multispecificity”embodiments of the “triple F” format, all with one monomer comprising anscFv and all of which have molecular weight differentials which can beexploited for purification of the heterodimers). FIG. 13A shows a“Fab-Fv” format with binding to two different antigens, with FIG. 13Bdepicting the “Fab-Fv” format with binding to a single antigen (e.g.bivalent binding to antigen 1). FIGS. 13C and 13D depicts a “Fv-Fab”format with similar bivalent or monovalent additional antigen binding.FIG. 13E depicts one monomer with a CH1-CL attached to the second scFv.

FIG. 14 depicts the sequence of optimized CD19 variable heavy and lightchains.

FIGS. 15A-15D depict four different triple F (“bottle opener”) sequencesfor anti-CD3-anti-CD19 heterodimeric antibodies. FIG. 15A shows: anoptimized heavy chain for CD19, including a pI engineered Fc utilizingpI variants (the (ISO−)) sequence and two variants for FcγR bindingablation (236R/328R; although as will be appreciated by those in theart, other “knock out” variants are discussed herein can also be used);an optimized anti-CD3 scFv in the context of the ISO(+RR) Fc domain withthe C220S background mutation and the same two variants for FcγR bindingablation (again, which can be exchanged for other ablation variants),and an optimized light chain. FIG. 15B shows the use of a differentlight chain. FIGS. 15C and 15D depict additional embodiments. The scFvsequences are shown in grey, and as discussed herein, other scFv linkerscan be utilized, particularly charged scFv linkers as shown in FIG. 7,although FIG. 15D shows the use of one of the charged scFv linkers.

FIG. 16 depicts the amino acid sequences for proof-of-concept systemused to determine the thermal stability of pI-altering isotypic constantregion variants of Example 5. Repair substitutions in ISO(−NKV) variantare underlined.

FIG. 17 depicts the differential scanning calorimetry (DSC) measurementsof thermal stability of pI-altering isotypic constant region variants.

FIG. 18 shows the amino acid sequences for proof-of-concept system usedto determine the heterodimer yield and thermal stability of pI-alteringisotypic constant region variants with added heterodimer-skewing Fcvariants (L368D/K370S-S364K/E357Q, underlined).

FIG. 19 shows an HPLC-CIEX measurement of heterodimer yield ofpI-altering isotypic constant region variants with addedheterodimer-skewing Fc variants.

FIG. 20 shows a differential scanning calorimetry (DSC) measurements ofthermal stability of pI-altering isotypic constant region variants withadded heterodimer-skewing Fc variants.

FIGS. 21A-21B depict the amino acid sequences for an anti-CD19 Fab xanti-CD3 scFv “triple F” embodiment, showing the HC-Fab sequence, theHC-scFv sequence, and the light chain. Amino acid substitutions made toallow for efficient purification of the desired “triple F” heterodimerover the undesired dual scFv-Fc and mAb homodimers are underlined.

FIG. 22 shows the amino acid sequences for the anti-CD38 Fab x anti-CD3scFv “triple F” embodiment, showing the HC-Fab sequence, the HC-scFvsequence, and the light chain. Amino acid substitutions made to allowfor efficient purification of the desired “triple F” heterodimer overthe undesired dual scFv-Fc and mAb homodimers are underlined.

FIGS. 23A-23B depict further amino acid sequences for embodiments of theanti-CD19 Fab x anti-CD3 scFv “triple F” bispecific, showing the HC-Fabsequence, the HC-scFv sequence, and the light chain for each bispecific.Amino acid substitutions made to “skew” the formation toward desired“triple F” heterodimer over undesired dual scFv-Fc and mAb homodimersare underlined.

FIG. 24 shows the literature pIs of the 20 amino acids. It should benoted that the listed pIs are calculated as free amino acids; the actualpI of any side chain in the context of a protein is different, and thusthis list is used to show pI trends and not absolute numbers for thepurposes of the invention.

FIG. 25A-25F is a data table of exemplary pI-engineered variants asshown below that find use in the Triple F format herein. It should benoted that FIG. 25 has SEQ ID NO:s that are associated with the sequencelisting filed in U.S. Ser. No. 13/648,951, and are hereby expresslyincorporated by reference.

XenP# the internal reference number Name (HC) heavy chain sequencedesignation SEQ ID NO corresponding SEQ ID NO of the heavy chainsequence (HC) Name (LC) light chain sequence designation SEQ ID NOcorresponding SEQ ID NO of the light chain sequence (LC) Calc. pIcalculated pI value for the entire antibody sequence, including heavyand light chain Fv + constant domains, with the Fv of bevacizumab andthe constant domains as defined in the table #KR number of Lys or Argresidues in IgG1 with the Fv of bevacizumab and the constant domains asdefined in the table Delta KR change in the number of Lys or Argresidues relative to (vs. WT) IgG1 wild-type sequence of bevacizumab #DEnumber of Asp or Glu residues in IgG1 with the Fv of bevacizumab and theconstant domains as defined in the table Delta DE change in the numberof Asp or Glu acid residues relative (vs. WT) to IgG1 wild-type sequenceof bevacizumab Charge state derived from the total number of Lys and Argminus the total number of Asp and Glu residues, assuming a pH of 7 # HCnumber of mutations in the heavy chain constant domain as Mutationscompared to IgG1 vs IgG1 # LC number of mutations in the light chainconstant domain as Mutations compared to IgG1 vs IgG1 Total # of totalnumber of mutations in the heavy chain and light Mutations chainconstant domains as compared to IgG1

FIGS. 26A-26B is an additional list of potential heterodimerizationvariants for use in the present invention, including isotypic variants.

FIG. 27 depicts the remarkable “skew” towards heterodimerization usingvariants of the invention. Heterodimerization of over 95% wasaccomplished using one monomer with L368E/K370T and the other with S364Kas compared to the same molecule without the Fc variants.

FIG. 28 depicts a matrix of possible combinations of heterodimerizationformats, heterodimerization variants (separated into pI variants andsteric variants (which includes charge pair variants), Fc variants, FcRnvariants and combinations. Legend A are suitable FcRn variants: 434A,434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,436V/428L, 252Y, 252Y/254T/256E and 259I/308F/428L. That is, the TripleF format of FIG. 1B can have any of these FcRn variants on either orboth monomer sequences. For clarity, as each heavy chain is different,FcRn variants (as well as the Fc variants) can reside on one or bothmonomers. Legend B are suitable Fc variants: 236A, 239D, 239E, 332E,332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y,239D, 332E/330L, 236R, 328R, 236R/328R, 236N/267E, 243L, 298A and 299T.(Note, additional suitable Fc variants are found in FIG. 41 of US2006/0024298, the figure and legend of which are hereby incorporated byreference in their entirety). In some cases as described herein, “knockout” or “ablation” variants are used such as depicted in FIG. 35, andthey are included in the definition of Fc variants. As for FcRnvariants, the Fc variants can reside on either strand. Legend C aresuitable pI variants, and these, for brevity are imported from FIG. 23,again with the understanding that there is a “strandedness” to pIvariants. Legend D are suitable steric variants (including charge pairvariants); again, for brevity are imported from FIGS. 9, 32 and 33,again with the understanding that there is a “strandedness” to stericvariants. Legend E reflects the following possible combinations, again,with each variant being independently and optionally combined from theappropriate source Legend: 1) pI variants plus FcRn variants; 2) pIvariants plus Fc variants; 3) pI variants plus FcRn variants plus Fcvariants; 4) steric variants plus FcRn variants; 5) steric variants plusFc variants; 6) steric variants plus FcRn variants plus Fc variants; 7)pI variants plus steric variants plus FcRn variants; 8) pI variants plussteric variants plus Fc variants; 9) pI variants plus steric variantsplus FcRn variants plus Fc variants; and 10) pI variants plus stericvariants. Note any or all of these combinations can optionally includeor exclude the knock out/ablation variants in either or both monomers.

FIGS. 29A-29 B depict all possible isotypic pI variants. As is outlinedherein, these can be independently and optionally combined with allother heterodimerization, retaining the appropriate “strandedness” (forboth pI and steric variants).

FIGS. 30A-30C depicts a number of pI variants that can additionally finduse in the present invention, which is a list of all possible reduced pIvariants created from isotypic substitutions of IgG1-4. Shown are the pIvalues for the three expected species as well as the average delta pIbetween the heterodimer and the two homodimer species present when thevariant heavy chain is transfected with IgG1-WT heavy chain.

FIG. 31 is a list of all possible increased pI variants created fromisotypic substitutions of IgG1-4. Shown are the pI values for the threeexpected species as well as the average delta pI between the heterodimerand the two homodimer species present when the variant heavy chain istransfected with IgG1-WT heavy chain.

FIGS. 32A-32B depict a number of preferred heterodimerization variants.Again, all of these can be independently and optionally combined withother heterodimerization variants, as long as “strandedness” ispreserved as needed.

FIG. 33 depicts some particularly useful pI isotypic variants and pairs.As is described herein, the figure depicts engineering of both monomers(one to more positive, one to more negative); however, it is alsopossible to engineer just one monomer.

FIGS. 34A-34C. FIG. 34A depicts two different Triple F embodiments.FIGS. 34B and 34C show the sequences of the Triple F embodiment of FIG.34A.

FIG. 35 depicts a number of suitable “knock out” (“KO”) variants toreduce binding to some or all of the FcγR receptors. As is true for manyif not all variants herein, these KO variants can be independently andoptionally combined, both within the set described in FIG. 35 and withany heterodimerization variants outlined herein, including steric and pIvariants. For example, E233P/L234V/L235A/G236del can be combined withany other single or double variant from the list. In addition, while itis preferred in some embodiments that both monomers contain the same KOvariants, it is possible to combine different KO variants on differentmonomers, as well as have only one monomer comprise the KO variant(s).Reference is also made to the Figures and Legends of U.S. Ser. No.61/913,870, all of which is expressly incorporated by reference in itsentirety as it relates to “knock out” or “ablation” variants.

FIG. 36 List of engineered heterodimer-skewing Fc variants withheterodimer yields (determined by HPLC-CIEX) and thermal stabilities(determined by DSC). Not determined thermal stability is denoted by“n.d.”.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1-76 of U.S. Ser. No. 61/593,846 and the associated legends anddiscussion in the specification are hereby incorporated by reference.

U.S. Ser. Nos. 61/778,157 and 61/818,410 are specifically and completelyincorporated by reference in its entirety, particularly the Figures andLegends therein.

I. Overview

The present invention is directed to novel constructs to providebispecific antibodies (or, as discussed below, trispecific ortetraspecific antibodies can also be made). An ongoing problem inantibody technologies is the desire for “bispecific” (and/ormultispecific) antibodies that bind to two (or more) different antigenssimultaneously, in general thus allowing the different antigens to bebrought into proximity and resulting in new functionalities and newtherapies. In general, these antibodies are made by including genes foreach heavy and light chain into the host cells. This generally resultsin the formation of the desired heterodimer (A-B), as well as the twohomodimers (A-A and B-B). However, a major obstacle in the formation ofmultispecific antibodies is the difficulty in purifying theheterodimeric antibodies away from the homodimeric antibodies and/orbiasing the formation of the heterodimer over the formation of thehomodimers.

The present invention is generally directed to the creation ofheterodimeric proteins such as antibodies that can co-engage antigens inseveral ways, relying on amino acid variants in the constant regionsthat are different on each chain to promote heterodimeric formationand/or allow for ease of purification of heterodimers over thehomodimers.

Thus, the present invention is directed to novel immunoglobulincompositions that co-engage at least a first and a second antigen. Firstand second antigens of the invention are herein referred to as antigen-1and antigen-2 respectively. One heavy chain of the antibody contains ansingle chain Fv (“scFv”, as defined below) and the other heavy chain isa “regular” FAb format, comprising a variable heavy chain and a lightchain. This structure is sometimes referred to herein as “triple F”format (scFv-FAb-Fc) or the “bottle-opener” format, due to a roughvisual similarity to a bottle-opener (see Figure). The two chains arebrought together by the use of amino acid variants in the constantregions (e.g. the Fc domain and/or the hinge region) that promote theformation of heterodimeric antibodies as is described more fully below.

There are several distinct advantages to the present “triple F” format.As is known in the art, antibody analogs relying on two scFv constructsoften have stability and aggregation problems, which can be alleviatedin the present invention by the addition of a “regular” heavy and lightchain pairing. In addition, as opposed to formats that rely on two heavychains and two light chains, there is no issue with the incorrectpairing of heavy and light chains (e.g. heavy 1 pairing with light 2,etc.)

There are a number of mechanisms that can be used to generate theheterodimers of the present invention. In addition, as will beappreciated by those in the art and described more fully below, thesemechanisms can be combined to ensure high heterodimerization.

One mechanism is generally referred to in the art as “knobs and holes”(“KIH”), or sometimes herein as “skew” variants, referring to amino acidengineering that creates steric influences to favor heterodimericformation and disfavor homodimeric formation can also optionally beused; this is sometimes referred to as “knobs and holes”, as describedin U.S. Ser. No. 61/596,846, Ridgway et al., Protein Engineering9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Pat. No.8,216,805, all of which are hereby incorporated by reference in theirentirety. The figures identify a number of “monomer A-monomer B” pairsthat rely on “knobs and holes”. In addition, as described in Merchant etal., Nature Biotech. 16:677 (1998), these “knobs and hole” mutations canbe combined with disulfide bonds to skew formation toheterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R. (Notethe 220 mutation is to remove a cysteine no longer needed for heavy andlight chain disulfide formation, as more fully described below).

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric proteins; one relies on the useof pI variants, such that each monomer has a different pI, thus allowingthe isoelectric purification of A-A, A-B and B-B dimeric proteins.Alternatively, the “triple F” format also allows separation on the basisof size. As is further outlined below, it is also possible to “skew” theformation of heterodimers over homodimers, as is generally outlinedbelow. Thus, a combination of steric heterodimerization variants and pIor charge pair variants find particular use in the invention.Additionally, as more fully outlined below, the scFv monomer of theTriple F format can include charged scFv linkers (either positive ornegative), that give a further pI boost for purification purposes. Aswill be appreciated by those in the art, some Triple F formats areuseful with just charged scFv linkers and no additional pI adjustments,although the invention does provide the use of skew variants withcharged scFv linkers as well (and combinations of Fc, FcRn and KOvariants).

In the present invention that utilizes pI as a separation mechanism tocreate the heterodimeric Triple F format, amino acid variants can beintroduced into one or both of the monomer polypeptides; that is, the pIof one of the monomers (referred to herein for simplicity as “monomerA”) can be engineered away from monomer B, or both monomer A and Bchange be changed, with the pI of monomer A increasing and the pI ofmonomer B decreasing. As is outlined more fully below, the pI changes ofeither or both monomers can be done by removing or adding a chargedresidue (e.g. a neutral amino acid is replaced by a positively ornegatively charged amino acid residue, e.g. glycine to glutamic acid),changing a charged residue from positive or negative to the oppositecharge (aspartic acid to lysine) or changing a charged residue to aneutral residue (e.g. loss of a charge; lysine to serine). A number ofthese variants are shown in the Figures.

Accordingly, in this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseit's pI (wt A−+B or wt A−−B), or by increasing one region and decreasingthe other region (A+−B− or A−B+). It should be noted that in thisdiscussion it does not matter which monomer comprises the scFv and whichthe Fab.

Thus, in general, a component of the present invention are amino acidvariants in the constant regions of antibodies that are directed toaltering the isoelectric point (pI) of at least one, if not both, of themonomers of a dimeric protein to form “pI heterodimers” (when theprotein is an antibody, these are referred to as “pI antibodies”) byincorporating amino acid substitutions (“pI variants” or “pIsubstitutions”) into one or both of the monomers. As shown herein, theseparation of the heterodimers from the two homodimers can beaccomplished if the pIs of the two monomers differ by as little as 0.1pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use in thepresent invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the scFv and Fab of interest. Thatis, to determine which monomer to engineer or in which “direction” (e.g.more positive or more negative), the Fv sequences of the two targetantigens are calculated and a decision is made from there. As is knownin the art, different Fvs will have different starting pIs which areexploited in the present invention. In general, as outlined herein, thepIs are engineered to result in a total pI difference of each monomer ofat least about 0.1 logs, with 0.2 to 0.5 being preferred as outlinedherein.

Furthermore, as will be appreciated by those in the art and outlinedherein, heterodimers can be separated from homodimers on the basis ofsize. For example, as shown in FIG. 11, heterodimers with two scFvs(FIG. 1A) can be separated by those of the “triple F” format (FIG. 1B)and a bispecific mAb (FIG. 1C). This can be further exploited in highervalency with additional antigen binding sites being utilized. Forexample, as additionally shown, one monomer will have two Fab fragmentsand the other will have one scFv, resulting in a differential in sizeand thus molecular weight.

In addition, as will be appreciated by those in the art and outlinedherein, the format outlined herein can be expanded to providetrispecific and tetraspecific antibodies as well. In this embodiment,some variations of which are depicted in the Figures, it will berecognized that it is possible that some antigens are bound divalently(e.g. two antigen binding sites to a single antigen; for example, A andB could be part of a typical bivalent association and C and D can beoptionally present and optionally the same or different). As will beappreciated, any combination of Fab and scFvs can be utilized to achievethe desired result and combinations.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of the heavy chain(s), a more modularapproach to designing and purifying multispecific proteins, includingantibodies, is provided. Thus, in some embodiments, heterodimerizationvariants (including skew and purification heterodimerization variants)are not included in the variable regions, such that each individualantibody must be engineered. In addition, in some embodiments, thepossibility of immunogenicity resulting from the pI variants issignificantly reduced by importing pI variants from different IgGisotypes such that pI is changed without introducing significantimmunogenicity. Thus, an additional problem to be solved is theelucidation of low pI constant domains with high human sequence content,e.g. the minimization or avoidance of non-human residues at anyparticular position.

In one embodiment, the heterodimeric antibody provides for monovalentengagement of one antigen using a scFv and monovalent engagement of theother antigen using a FAb. As outlined below, this format can also bevaried; in some embodiments, there is monovalent engagement of threeantigens, divalent engagement of one antigen and monovalent engagementof a second antigen (e.g. A and C are to the same antigen and B is to adifferent antigen), etc.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition to all or part of a variant heavy constant domain, one orboth of the monomers may contain one or two fusion partners, such thatthe heterodimers form multivalent proteins. As is generally depicted inthe FIG. 64 of U.S. Ser. No. 13/648,951, hereby incorporated byreference with its accompanying legend, the fusion partners are depictedas A, B, C and D, with all combinations possible. In general, A, B, Cand D are selected such that the heterodimer is at least bispecific orbivalent in its ability to interact with additional proteins. In thecontext of the present “triple F” format, generally A and B are an scFvand a Fv (as will be appreciated, either monomer can contain the scFvand the other the Fv/Fab) and then optionally one or two additionalfusion partners.

Furthermore, as outlined herein, additional amino acid variants may beintroduced into the bispecific antibodies of the invention, to addadditional functionalities. For example, amino acid changes within theFc region can be added (either to one monomer or both) to facilitiateincreased ADCC or CDC (e.g. altered binding to Fcγ receptors); to allowor increase yield of the addition of toxins and drugs (e.g. for ADC), aswell as to increase binding to FcRn and/or increase serum half-life ofthe resulting molecules. As is further described herein and as will beappreciated by those in the art, any and all of the variants outlinedherein can be optionally and independently combined with other variants.

Similarly, another category of functional variants are “Fcγ ablationvariants” or “Fc knock out (FcKO or KO) variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific antibodies that bindCD3 monovalently and a tumor antigen on the other (e.g. CD19, her2/neu,etc.), it is generally desirable to ablate FcγRIIIa binding to eliminateor significantly reduce ADCC activity.

In addition, the invention provides novel humanized anti-CD3 sequences,including sets of CDRs, full variable light and heavy chains, as well asthe associated scFvs, which can optionally include charged scFv linkers.These optimized sequences can be used in other antibody formats.

Accordingly, the present invention provides novel constructs to producemultivalent antibodies.

II. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Variants ofparticular use when ablation variants (also sometimes referred to hereinas “FcγR ablation variants”, “Fc ablation variants”, “Fc knock outs”(“FcKO”) or “knock out” (“KO”) variants) are used are those depicted inFIG. 35.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233# or E233del designatesa deletion of glutamic acid at position 233. Additionally, EDA233- orEDA233# designates a deletion of the sequence GluAspAla that begins atposition 233. Similarly, some of the heterodimerization variants include“K447del”, meaning the lysine at position 447 has been deleted.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”. The proteinvariant sequence herein will preferably possess at least about 80%identity with a parent protein sequence, and most preferably at leastabout 90% identity, more preferably at least about 95-98-99% identity.Variant protein can refer to the variant protein itself, compositionscomprising the protein variant, or the DNA sequence that encodes it.Accordingly, by “antibody variant” or “variant antibody” as used hereinis meant an antibody that differs from a parent antibody by virtue of atleast one amino acid modification, “IgG variant” or “variant IgG” asused herein is meant an antibody that differs from a parent IgG (again,in many cases, from a human IgG sequence) by virtue of at least oneamino acid modification, and “immunoglobulin variant” or “variantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification. “Fc variant” or “variant Fc” as usedherein is meant a protein comprising an amino acid modification in an Fcdomain. The Fc variants of the present invention are defined accordingto the amino acid modifications that compose them. Thus, for example,N434S or 434S is an Fc variant with the substitution serine at position434 relative to the parent Fc polypeptide, wherein the numbering isaccording to the EU index. Likewise, M428L/N434S defines an Fc variantwith the substitutions M428L and N434S relative to the parent Fcpolypeptide. The identity of the WT amino acid may be unspecified, inwhich case the aforementioned variant is referred to as 428L/434S. It isnoted that the order in which substitutions are provided is arbitrary,that is to say that, for example, 428L/434S is the same Fc variant asM428L/N434S, and so on. For all positions discussed in the presentinvention that relate to antibodies, unless otherwise noted, amino acidposition numbering is according to the EU index. The EU index or EUindex as in Kabat or EU numbering scheme refers to the numbering of theEU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,hereby entirely incorporated by reference.) The modification can be anaddition, deletion, or substitution. Substitutions can include naturallyoccurring amino acids and, in some cases, synthetic amino acids.Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO 03/073238;US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, etal., (2002), PICAS United States of America 99:11020-11024; and, L.Wang, & P. G. Schultz, (2002), Chem. 1-10, all entirely incorporated byreference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fvfragment” or “Fv region” as used herein is meant a polypeptide thatcomprises the VL and VH domains of a single antibody.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification. “Isotypic” modifications refer tothe importation of one isotype amino acid at a position into thebackbone of a different isotype; for example, the importation of an IgG1amino acid into an IgG2 backbone at the same position.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcqammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life, are discussed in Legend A of FIG.24.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as a bindingmoiety to a target protein, as described herein).

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “strandedness” in the context of the monomers of the heterodimericproteins of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g. making the pI higher) then steric variants that are“charge pairs” that can be utilized as well do not interfere with the pIvariants, e.g. the charge variants that make a pI higher are put on thesame “strand” or “monomer” to preserve both functionalities.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. A wide number of suitable target antigens are described below.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V.kappa., V.lamda., and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

By “single chain variable fragment”, “scFv” or “single chain Fv” as iswell understood in the art, herein is meant a fusion protein of thevariable heavy and light chains of an antibody, usually linked with alinker peptide. Typical scFv linkers are well known in the art, aregenerally 10 to 25 amino acids in length and include glycines andserines.

By “charged scFv linker” herein is meant a scFv linker that utilizescharged amino acids for use in the creation and purification ofheterodimeric antibodies that include at least one scFv. Suitablecharged scFv linkers are shown in FIG. 7, although others can be used.In general, the charged scFv linkers for use in the present inventionhave a charge change from 3 to 8 (3, 4, 5, 6, 7 or 8 all being possible)as compared to the standard uncharged scFv linkers such as (GGGGS)₃₋₅sequences traditionally used (either negative or positive). As will beappreciated by those in the art, heterodimeric antibodies that utilizetwo scFvs can have one charged and one neutral linker (e.g. either apositively or negatively charged scFv linker) or two oppositely chargedscFv linkers (one positive and one negative).

Heterodimeric Proteins

The present invention is directed to the generation of multispecific,particularly bispecific binding proteins, and in particular,multispecific antibodies that have one monomer comprising an scFv andthe other an Fv.

Antibodies

The present invention relates to the generation of multispecificantibodies, generally therapeutic antibodies. As is discussed below, theterm “antibody” is used generally. Antibodies that find use in thepresent invention can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described below. In general, the term “antibody”includes any polypeptide that includes at least one constant domain,including, but not limited to, CH1, CH2, CH3 and CL.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Itshould be understood that therapeutic antibodies can also comprisehybrids of isotypes and/or subclasses. For example, as shown in USPublication 2009/0163699, incorporated by reference, the presentinvention covers pI engineering of IgG1/G2 hybrids.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) (e.g, Kabat et al.,supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

It should be noted that the sequences depicted herein start at the CH1region, position 118; the variable regions are not included except asnoted. For example, the first amino acid of SEQ ID NO: 2, whiledesignated as position“1” in the sequence listing, corresponds toposition 118 of the CH1 region, according to EU numbering.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or Cκ).

Another region of interest for additional substitutions, outlined below,is the Fc region. By “Fc” or “Fc region” or “Fc domain” as used hereinis meant the polypeptide comprising the constant region of an antibodyexcluding the first constant region immunoglobulin domain and in somecases, part of the hinge. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM, Fc may include the Jchain. For IgG, the Fc domain comprises immunoglobulin domains Cγ2 andCγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2(Cγ2). Although the boundaries of the Fc region may vary, the human IgGheavy chain Fc region is usually defined to include residues C226 orP230 to its carboxyl-terminus, wherein the numbering is according to theEU index as in Kabat. In some embodiments, as is more fully describedbelow, amino acid modifications are made to the Fc region, for exampleto alter binding to one or more FcγR receptors or to the FcRn receptor.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can include a variety of structures,including, but not limited to, antibody fragments, monoclonalantibodies, bispecific antibodies, minibodies, domain antibodies,synthetic antibodies (sometimes referred to herein as “antibodymimetics”), chimeric antibodies, humanized antibodies, antibody fusions(sometimes referred to as “antibody conjugates”), and fragments of each,respectively.

In one embodiment, the antibody is an antibody fragment, as long as itcontains at least one constant domain which can be engineered to produceheterodimers, such as pI engineering. Other antibody fragments that canbe used include fragments that contain one or more of the CH1, CH2, CH3,hinge and CL domains of the invention that have been pI engineered. Forexample, Fc fusions are fusions of the Fc region (CH2 and CH3,optionally with the hinge region) fused to another protein. A number ofFc fusions are known the art and can be improved by the addition of theheterodimerization variants of the invention. In the present case,antibody fusions can be made comprising CH1; CH1, CH2 and CH3; CH2; CH3;CH2 and CH3; CH1 and CH3, any or all of which can be made optionallywith the hinge region, utilizing any combination of heterodimerizationvariants described herein.

In some embodiments of the present invention, one monomer comprises aheavy chain comprises a scFV linked to an Fc domain, and the othermonomer comprises a heavy chain comprising a Fab linked to an Fc domain,e.g. a “typical” heavy chain, and a light chain. By “Fab” or “Fabregion” as used herein is meant the polypeptide that comprises the VH,CH1, VL, and CL immunoglobulin domains. Fab may refer to this region inisolation, or this region in the context of a full length antibody,antibody fragment or Fab fusion protein. By “Fv” or “Fv fragment” or “Fvregion” as used herein is meant a polypeptide that comprises the VL andVH domains of a single antibody.

Chimeric and Humanized Antibodies

In some embodiments, the antibody can be a mixture from differentspecies, e.g. a chimeric antibody and/or a humanized antibody. Ingeneral, both “chimeric antibodies” and “humanized antibodies” refer toantibodies that combine regions from more than one species. For example,“chimeric antibodies” traditionally comprise variable region(s) from amouse (or rat, in some cases) and the constant region(s) from a human.“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. No. 5,530,101;U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat.No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, allentirely incorporated by reference). The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system. Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference). Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference.Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference. In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

Multispecific Antibody Constructs

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention takeon a number variety of configurations, with a preferred embodiment shownin FIG. 1B as a “triple F” construct.

Heterodimeric Heavy Chain Constant Regions

Accordingly, the present invention provides heterodimeric proteins basedon the use of monomers containing variant heavy chain constant regionsas a first domain. By “monomer” herein is meant one half of theheterodimeric protein. It should be noted that traditional antibodiesare actually tetrameric (two heavy chains and two light chains). In thecontext of the present invention, one pair of heavy-light chains (ifapplicable, e.g. if the monomer comprises an Fab) is considered a“monomer”. Similarly, a heavy chain region comprising the scFv isconsidered a monomer. In the case where an Fv region is one fusionpartner (e.g. heavy and light chain) and a non-antibody protein isanother fusion partner, each “half” is considered a monomer.Essentially, each monomer comprises sufficient heavy chain constantregion to allow heterodimerization engineering, whether that be all theconstant region, e.g. Ch1-hinge-CH2-CH3, the Fc region (CH2-CH3), orjust the CH3 domain.

The variant heavy chain constant regions can comprise all or part of theheavy chain constant region, including the full length construct,CH1-hinge-CH2-CH3, or portions thereof, including for example CH2-CH3 orCH3 alone. In addition, the heavy chain region of each monomer can bethe same backbone (CH1-hinge-CH2-CH3 or CH2-CH3) or different. N- andC-terminal truncations and additions are also included within thedefinition; for example, some pI variants include the addition ofcharged amino acids to the C-terminus of the heavy chain domain.

Thus, in general, one monomer of the present “triple F” construct is ascFv region-hinge-Fc domain) and the other is (VH-CH1-hinge-CH2-CH3 plusassociated light chain), with heterodimerization variants, includingsteric and pI variants, Fc and FcRn variants, and additional antigenbinding domains (with optional linkers) included in these regions.

In addition to the heterodimerization variants (e.g. steric and pIvariants) outlined herein, the heavy chain regions may also containadditional amino acid substitutions, including changes for altering FcγRand FcRn binding as discussed below.

In addition, some monomers can utilize linkers between the variant heavychain constant region and the fusion partner. For the scFv portion ofthe “bottle-opener”, standard linkers as are known in the art can beused. In the case where additional fusion partners are made (e.g. FIG.64 of U.S. Ser. No. 13/648,951), traditional peptide linkers can beused, including flexible linkers of glycine and serine. In some cases,the linkers for use as components of the monomer are different fromthose defined below for the ADC constructs, and are in many embodimentsnot cleavable linkers (such as those susceptible to proteases), althoughcleavable linkers may find use in some embodiments.

Alternatively, depending on the antibody format, one or more chargedscFv linkers can be utilized as outlined herein. In Triple F format, onecharged scFv linker is used. As noted herein, depending on the inherentpI of the scFv for the target antigen and the inherent pI of the Fab ofthe other target antigen, the charged scFv linker can either be positiveor negative. In dual scFv formats, either a single charged scFv linkeris used on one monomer (again, either positive or negative) or both (onepositive and one negative). In this embodiment, the charge of each ofthe two linkers need not be the same (e.g. +3 for one and −4 for theother, etc.).

The heterodimerization variants include a number of different types ofvariants, including, but not limited to, steric variants (includingcharge variants) and pI variants, that can be optionally andindependently combined with any other variants. In these embodiments, itis important to match “monomer A” with “monomer B”; that is, if aheterodimeric protein relies on both steric variants and pI variants,these need to be correctly matched to each monomer: e.g. the set ofsteric variants that work (1 set on monomer A, 1 set on monomer B) iscombined with pI variant sets (1 set on monomer A, 1 set on monomer B),such that the variants on each monomer are designed to achieve thedesired function. In the case for example where steric variants may alsochange the charge, the correct sets have to be matched to the correctmonomer.

It is important to note that the heterodimerization variants outlinedherein (for example, including but not limited to those variants shownin FIGS. 9, 26, 29, 30, 31 and 32), can be optionally and independentlycombined with any other variants, and on any other monomer. Thus, forexample, pI variants for monomer 1 from one figure can be added to otherheterodimerization variants for monomer 1 in a different figure or frommonomer 2. That is, what is important for the heterodimerization is thatthere are “sets” of variants, one set for one monomer and one set forthe other. Whether these are combined from the FIGS. 1 to 1 (e.g.monomer 1 listings can go together) or switched (monomer 1 pI variantswith monomer 2 steric variants) is irrelevant. However, as noted herein,“strandedness” should be preserved when combinations are made asoutlined above such that heterodimerization is favored; e.g. chargevariants that increase pI should be used with increased pI variantsand/or an scFv linker with increase pI, etc. Furthermore, for theadditional Fc variants (such as for FcγR binding, FcRn binding, ablationvariants etc.), either monomer, or both monomers, can include any of thelisted variants, independently and optionally. In some cases, bothmonomers have the additional variants and in some only one monomer hasthe additional variants, or they can be combined.

Heterodimerization Variants

The present invention provides multispecific antibody formats, on a“triple F” or “bottle opener” scaffold as depicted in FIG. 11B, forexample.

Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are shown in theFigures

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. FIGS. 4 and 5, further described below,identifies a number of “monomer A-monomer B” pairs that rely on “knobsand holes”. In addition, as described in Merchant et al., NatureBiotech. 16:677 (1998), these “knobs and hole” mutations can be combinedwith disulfide bonds to skew formation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, variants resulting ingreater than 75% heterodimerization in the Figures such asD221E/P228E/L368E paired with D221R/P228R/K409R (e.g. these are “monomercorresponding sets) and C220E/P228E/368E paired withC220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend of which are incorporatedexpressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any heterodimerizationvariants including pI variants (or other variants such as Fc variants,FcRn variants, ablation variants, etc.) into one or both monomers.

pI (Isoelectric Point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in the Figures.

Heavy Chain Acidic pI Changes

Accordingly, when one monomer comprising a variant heavy chain constantdomain is to be made more positive (e.g. lower the pI), one or more ofthe following substitutions can be made: S119E, K133E, K133Q, T164E,K205E, K205Q, N208D, K210E, K210Q, K274E, K320E, K322E, K326E, K334E,R355E, K392E, a deletion of K447, adding peptide DEDE at the c-terminus,G137E, N203D, K274Q, R355Q, K392N and Q419E. As outlined herein andshown in the figures, these changes are shown relative to IgG1, but allisotypes can be altered this way, as well as isotype hybrids.

In the case where the heavy chain constant domain is from IgG2-4, R133Eand R133Q can also be used.

Basic pI Changes

Accordingly, when one monomer comprising a variant heavy chain constantdomain is to be made more negative (e.g. increase the pI), one or moreof the following substitutions can be made: Q196K, P217R, P228R, N276Kand H435R. As outlined herein and shown in the figures, these changesare shown relative to IgG1, but all isotypes can be altered this way, aswell as isotype hybrids.

Antibody Heterodimers Light Chain Variants

In the case of antibody based heterodimers, e.g. where at least one ofthe monomers comprises a light chain in addition to the heavy chaindomain, pI variants can also be made in the light chain. Amino acidsubstitutions for lowering the pI of the light chain include, but arenot limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E,K207E and adding peptide DEDE at the c-terminus of the light chain.Changes in this category based on the constant lambda light chaininclude one or more substitutions at R108Q, Q124E, K126Q, N138D, K145Tand Q199E. In addition, increasing the pI of the light chains can alsobe done.

Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. That is, IgG1 is acommon isotype for therapeutic antibodies for a variety of reasons,including high effector function. However, the heavy constant region ofIgG1 has a higher pI than that of IgG2 (8.10 versus 7.31). Byintroducing IgG2 residues at particular positions into the IgG1backbone, the pI of the resulting monomer is lowered (or increased) andadditionally exhibits longer serum half-life. For example, IgG1 has aglycine (pI 5.97) at position 137, and IgG2 has a glutamic acid (pI3.22); importing the glutamic acid will affect the pI of the resultingprotein. As is described below, a number of amino acid substitutions aregenerally required to significant affect the pI of the variant antibody.However, it should be noted as discussed below that even changes in IgG2molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the Figures.Alternatively, the pI of each monomer can be compared. Similarly, thepIs of the “starting” variable regions (e.g. either scFv or Fab) arecalculated to inform which monomer will be engineered in whichdirection.

pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall'Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half life as wild-type Fc (Dall'Acqua et al.2002, J. Immunol. 169:5171-5180, entirely incorporated by reference).The increased affinity of Fc for FcRn at pH 7.4 is thought to forbid therelease of the Fc back into the blood. Therefore, the Fc mutations thatwill increase Fc's half-life in vivo will ideally increase FcRn bindingat the lower pH while still allowing release of Fc at higher pH. Theamino acid histidine changes its charge state in the pH range of 6.0 to7.4. Therefore, it is not surprising to find His residues at importantpositions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

pI variants that find use in this embodiment, as well as their use forpurification optimization, are disclosed in the Figures.

Combination of Heterodimeric Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants can be optionally and independently combinedin any way, as long as they retain their “strandedness” or “monomerpartition”. In addition, all of these variants can be combined into anyof the hterodimerization formats. See FIG. 28 and its legend.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

Target Antigens

The immunoglobulins of the invention may target virtually any antigens.The “triple F” format is particularly beneficial for targeting two (ormore) distinct antigens. (As outlined herein, this targeting can be anycombination of monovalent and divalent binding, depending on theformat). Thus the immunoglobulins herein preferably co-engage two targetantigens, although in some cases, three or four antigens can bemonovalently engaged. Each monomer's specificity can be selected fromthe lists below. While the triple F immunoglobulins described herein areparticularly beneficial for targeting distinct antigens, in some casesit may be beneficial to target only one antigen. That is, each monomermay have specificity for the same antigen.

Particular suitable applications of the immunoglobulins herein areco-target pairs for which it is beneficial or critical to engage eachtarget antigen monovalently. Such antigens may be, for example, immunereceptors that are activated upon immune complexation. Cellularactivation of many immune receptors occurs only by cross-linking,acheived typically by antibody/antigen immune complexes, or via effectorcell to target cell engagement. For some immune receptors, for examplethe CD3 signaling receptor on T cells, activation only upon engagementwith co-engaged target is critical, as nonspecific cross-linking in aclinical setting can elicit a cytokine storm and toxicity.Therapeutically, by engaging such antigens monovalently rather thanmultivalently, using the immunoglobulins herein, such activation occursonly in response to cross-linking only in the microenvironment of theprimary target antigen. The ability to target two different antigenswith different valencies is a novel and useful aspect of the presentinvention. Examples of target antigens for which it may betherapeutically beneficial or necessary to co-engage monovalentlyinclude but are not limited to immune activating receptors such as CD3,FcγRs, toll-like receptors (TLRs) such as TLR4 and TLR9, cytokine,chemokine, cytokine receptors, and chemokine receptors. In manyembodiments, one of the antigen binding sites binds to CD3, and in someembodiments it is the scFv-containing monomer.

Virtually any antigen may be targeted by the immunoglobulins herein,including but not limited to proteins, subunits, domains, motifs, and/orepitopes belonging to the following list of target antigens, whichincludes both soluble factors such as cytokines and membrane-boundfactors, including transmembrane receptors: 17-IA, 4-1BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE,ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, ActivinRIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RITA, Activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAMS, ADAMTS,ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxinl, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, MCSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Mucl), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP),P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, 5100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEMS, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand,TL2), TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

Exemplary antigens that may be targeted specifically by theimmunoglobulins of the invention include but are not limited to: CD20,CD19, Her2, EGFR, EpCAM, CD3, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb(CD32b), FcγRI (CD64), Toll-like receptors (TLRs) such as TLR4 and TLR9,cytokines such as IL-2, IL-5, IL-13, IL-12, IL-23, and TNFα, cytokinereceptors such as IL-2R, chemokines, chemokine receptors, growth factorssuch as VEGF and HGF, and the like. To form the multispecific antibodiesof the invention, antibodies to any combination of these antigens can bemade; that is, each of these antigens can be optionally andindependently included or excluded from a multispecific antibodyaccording to the present invention.

Particularly preferred combinations for bispecific antibodies are anantigen-binding domain to CD3 and an antigen binding domain to CD19; anantigen-binding domain to CD3 and an antigen binding domain to CD33; anantigen-binding domain to CD3 and an antigen binding domain to CD 38.Again, in many embodiments, the CD3 binding domain is the scFv, havingan exemplary sequence as depicted in the Figures and/or CD3 CDRs asoutlined.

The choice of suitable target antigens and co-targets depends on thedesired therapeutic application. Some targets that have provenespecially amenable to antibody therapy are those with signalingfunctions. Other therapeutic antibodies exert their effects by blockingsignaling of the receptor by inhibiting the binding between a receptorand its cognate ligand. Another mechanism of action of therapeuticantibodies is to cause receptor down regulation. Other antibodies do notwork by signaling through their target antigen. The choice of co-targetswill depend on the detailed biology underlying the pathology of theindication that is being treated.

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (Weiner et al., 2010, Nature Reviews Immunology10:317-327; Reichert et al., 2005, Nature Biotechnology 23[9]:1073-1078;herein expressly incorporated by reference). For anti-cancer treatmentit may be desirable to target one antigen (antigen-1) whose expressionis restricted to the cancerous cells while co-targeting a second antigen(antigen-2) that mediates some immunulogical killing activity. For othertreatments it may be beneficial to co-target two antigens, for exampletwo angiogenic factors or two growth factors, that are each known toplay some role in proliferation of the tumor. Exemplary co-targets foroncology include but are not limited to HGF and VEGF, IGF-1R and VEGF,Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 andFcγRIIIa, CD20 and FcγRIIIa, Her2 and FcγRIIIa. An immunoglobulin of theinvention may be capable of binding VEGF and phosphatidylserine; VEGFand ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1;VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR andCDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20;CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 andCD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF andphosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and Her-2;c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20;IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 andVEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF;PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2;PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON andMSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR;VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4;VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4;CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 andCD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40;CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 andDR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 andCD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52;CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 andVEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, andEGFR and DLL4. Other target combinations include one or more members ofthe EGF/erb-2/erb-3 family.

Other targets (one or more) involved in oncological diseases that theimmunoglobulins herein may bind include, but are not limited to thoseselected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8,BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1,FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2,FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2,TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2,CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4,CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3,IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL,TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1,IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3,NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR112,NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2,NR5A1, NR5A2, NR6 μl, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10,FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2,IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10,KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18,CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164,COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1,CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3,FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A,IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID,PR1, PSCA, SLC2A2, SLC33 μl, SLC43 μl, STEAP, STEAP2, TPM1, TPM2, TRPC6,ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR,LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB 1, VEGF, VEGFC, ANGPTL3, BAI1,COL4A3, IL8, LAMA5, NRP1, NRP2, STAB 1, ANGPTL4, PECAM1, PF4, PROK2,SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2,EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2,TGFBR1, CCL2, CDH5, COL1A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B(p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin), CTSB(cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3,GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130),ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),NGFB (GF), NGFR, NME1 (M23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin),SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6(Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7(claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin),GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin),KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type IIkeratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2(COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1(mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2,THBS4, and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4,phophatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD80, CD55,CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL,VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2,NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1,PSMA, PSCA, SCD1, and CD59. To form the bispecific or trispecificantibodies of the invention, antibodies to any combination of theseantigens can be made; that is, each of these antigens can be optionallyand independently included or excluded from a multispecific antibodyaccording to the present invention.

Monoclonal antibody therapy has become an important therapeutic modalityfor treating autoimmune and inflammatory disorders (Chan & Carter, 2010,Nature Reviews Immunology 10:301-316; Reichert et al., 2005, NatureBiotechnology 23[9]:1073-1078; herein expressly incorporated byreference). Many proteins have been implicated in general autoimmune andinflammatory responses, and thus may be targeted by the immunogloublinsof the invention. Autoimmune and inflammatory targets include but arenot limited to C5, CCL1 (1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15(MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2(mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b),CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11(1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5(ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1,CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1(CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6,IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1(endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2,IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A,CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1,IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5,TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z,CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44,HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13,CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1,CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12,CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A,BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118,FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2,IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7,IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL12RA, IL12A,IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16,IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R,LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1,TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A,TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4,TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

Exemplary co-targets for autoimmune and inflammatory disorders includebut are not limited to IL-1 and TNFalpha, IL-6 and TNFalpha, IL-6 andIL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13,IL-9 and IL-13, CD19 and FcγRIIb, and CD79 and FcγRIIb.

Immunglobulins of the invention with specificity for the following pairsof targets to treat inflammatory disease are contemplated: TNF andIL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF andalphaVbeta3; TNF and NGF; TNF and IL-23p19; TNF and IL-6; TNF and SOST;TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE andIL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 andIL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R andRANKL; IL-17A and IL-1beta; IL-1beta and RANKL; IL-1beta and VEGF; RANKLand CD-20; IL-1alpha and IL-1beta; IL-1alpha and IL-1beta.

Pairs of targets that the immunoglobulins described herein can bind andbe useful to treat asthma may be determined. In an embodiment, suchtargets include, but are not limited to, IL-13 and IL-1beta, sinceIL-1beta is also implicated in inflammatory response in asthma; IL-13and cytokines and chemokines that are involved in inflammation, such asIL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHRagonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13and ADAMS. The immunoglobulins herein may have specifity for one or moretargets involved in asthma selected from the group consisting of CSF1(MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG, histamineand histamine receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8,IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19,KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2,IL13RA1, IL13RA2, IL18R1, TSLP, CCLi, CCL2, CCL3, CCL4, CCL5, CCL7,CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1,CXCL2, CXCL3, XCLi, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1,GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6, TBX21, TGFB1, TNF, TNFSF6,YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase. To formthe bispecific or trispecific antibodies of the invention, antibodies toany combination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

Pairs of targets involved in rheumatoid arthritis (RA) may beco-targeted by the invention, including but not limited to TNF andIL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1beta; TNF and MIF; TNFand IL-17; and TNF and IL-15.

Antigens that may be targeted in order to treat systemic lupuserythematosus (SLE) by the immunoglobulins herein include but are notlimited to CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2,IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A,HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, CD81, IFNB1, IL10, TNFRSF5,TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9,IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80,CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72,CD74, CD79A, CD79B, CR2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC,CHSTIO, HLA-A, HLA-DRA, and NT5E; CTLA4, B7.1, B7.2, BlyS, BAFF, C5,IL-4, IL-6, IL-10, IFN-α, and TNF-α. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

The immunoglobulins herein may target antigens for the treatment ofmultiple sclerosis (MS), including but not limited to IL-12, TWEAK,IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200,IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An embodiment includesco-engagement of anti-IL-12 and TWEAK for the treatment of MS.

One aspect of the invention pertains to immunoglobulins capable ofbinding one or more targets involved in sepsis, in an embodiment twotargets, selected from the group consisting TNF, IL-1, MIF, IL-6, IL-8,IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissuefactor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFκB1, PROC,TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFκB1, PTAFR, TLR2, TLR4, GPR44,HMOX1, midkine, IRAK1, NFκB2, SERPINA1, SERPINE1, and TREM1. To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

In some cases, immunoglobulins herein may be directed against antigensfor the treatment of infectious diseases.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10-4 M, at least about 10-5 M, at least about10-6 M, at least about 10-7 M, at least about 10-8 M, at least about10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, atleast about 10-12 M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

Modified Antibodies

In addition to the modifications outlined above, other modifications canbe made. For example, the molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996, Nature Biotech. 14:1239-1245, entirelyincorporated by reference). In addition, there are a variety of covalentmodifications of antibodies that can be made as outlined below.

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesmay also be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

In addition, modifications at cysteines are particularly useful inantibody-drug conjugate (ADC) applications, further described below. Insome embodiments, the constant region of the antibodies can beengineered to contain one or more cysteines that are particularly “thiolreactive”, so as to allow more specific and controlled placement of thedrug moiety. See for example U.S. Pat. No. 7,521,541, incorporated byreference in its entirety herein.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125I or 131I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods, in addition to methods described below. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such ascynomolgusogen bromide-activated carbohydrates and the reactivesubstrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; and 4,330,440, all entirely incorporated byreference, are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983],entirely incorporated by reference), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

In addition, as will be appreciated by those in the art, labels(including fluorescent, enzymatic, magnetic, radioactive, etc. can allbe added to the antibodies (as well as the other compositions of theinvention).

Glycosylation

Another type of covalent modification is alterations in glycosylation.In another embodiment, the antibodies disclosed herein can be modifiedto include one or more engineered glycoforms. By “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to the antibody, wherein said carbohydrate composition differschemically from that of a parent antibody. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. A preferred form of engineered glycoformis afucosylation, which has been shown to be correlated to an increasein ADCC function, presumably through tighter binding to the FcγRIIIareceptor. In this context, “afucosylation” means that the majority ofthe antibody produced in the host cells is substantially devoid offucose, e.g. 90-95-98% of the generated antibodies do not haveappreciable fucose as a component of the carbohydrate moiety of theantibody (generally attached at N297 in the Fc region). Definedfunctionally, afucosylated antibodies generally exhibit at least a 50%or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zurich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells, by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. For example,the “sugar engineered antibody” or “SEA technology” of Seattle Geneticsfunctions by adding modified saccharides that inhibit fucosylationduring production; see for example 20090317869, hereby incorporated byreference in its entirety. Engineered glycoform typically refers to thedifferent carbohydrate or oligosaccharide; thus an antibody can includean engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal. Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

Additional Fc Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγRreceptors, altered binding to FcRn receptors, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants

FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors.Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto Fc□RIIIa generally results in increased ADCC (antibody dependentcell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances. Amino acid substitutions thatfind use in the present invention include those listed in U.S. Ser. No.11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287, 11/396,495,11/538,406, all of which are expressly incorporated herein by referencein their entirety and specifically for the variants disclosed therein.Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L and 299T.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 428L,308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428Land 259I/308F/428L.

Fc Ablation Variants

Additional variants which find use in the present invention are thosethat ablate (e.g. reduce or eliminate) binding to Fcγ receptors. Thiscan be desirable to reduce the potential mechanisms of action (e.g.reduce ADCC activity) of the heterodimeric antibodies of the invention.A number of suitable Fc ablation variants are depicted in FIG. 35, andcan be optionally and independently included or excluded in combinationwith any other heterodimerization variants, including pI and stericvariants.

Linkers

The present invention optionally provides linkers as needed, for examplein the addition of additional antigen binding sites, as depicted forexample in FIGS. 11, 12 and 13, where “the other end” of the moleculecontains additional antigen binding components. In addition, as outlinedbelow, linkers are optionally also used in antibody drug conjugate (ADC)systems. When used to join the components of the central mAb-Fvconstructs, the linker is generally a polypeptide comprising two or moreamino acid residues joined by peptide bonds and are used to link one ormore of the components of the present invention. Such linkerpolypeptides are well known in the art (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123). A variety of linkers may find use in someembodiments described herein. As will be appreciated by those in theart, there are at least three different linker types used in the presentinvention.

“Linker” herein is also referred to as “linker sequence”, “spacer”,“tethering sequence” or grammatical equivalents thereof. Homo-orhetero-bifunctional linkers as are well known (see, 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated entirely by reference). (Note the distinction betweengeneric “linkers” and “scFv linkers and “charged scFv linkers”). Anumber of strategies may be used to covalently link molecules together.These include, but are not limited to polypeptide linkages between N-and C-termini of proteins or protein domains, linkage via disulfidebonds, and linkage via chemical cross-linking reagents. In one aspect ofthis embodiment, the linker is a peptide bond, generated by recombinanttechniques or peptide synthesis. The linker peptide may predominantlyinclude the following amino acid residues: Gly, Ser, Ala, or Thr. Thelinker peptide should have a length that is adequate to link twomolecules in such a way that they assume the correct conformationrelative to one another so that they retain the desired activity. In oneembodiment, the linker is from about 1 to 50 amino acids in length,preferably about 1 to 30 amino acids in length. In one embodiment,linkers of 1 to 20 amino acids in length may be used. Useful linkersinclude glycine-serine polymers, including for example (GS)n, (GSGGS)n,(GGGGS)n, and (GGGS)n, where n is an integer of at least one,glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers. Alternatively, a variety of nonproteinaceous polymers,including but not limited to polyethylene glycol (PEG), polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol, may find use as linkers, that is may find use aslinkers.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cκ or Cλ. Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g. TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

Antibody-Drug Conjugates

In some embodiments, the multispecific antibodies of the invention areconjugated with drugs to form antibody-drug conjugates (ADCs). Ingeneral, ADCs are used in oncology applications, where the use ofantibody-drug conjugates for the local delivery of cytotoxic orcytostatic agents allows for the targeted delivery of the drug moiety totumors, which can allow higher efficacy, lower toxicity, etc. Anoverview of this technology is provided in Ducry et al., BioconjugateChem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) andSenter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which arehereby incorporated by reference in their entirety.

Thus the invention provides multispecific antibodies conjugated todrugs. Generally, conjugation is done by covalent attachment to theantibody, as further described below, and generally relies on a linker,often a peptide linkage (which, as described below, may be designed tobe sensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides multispecific antibodies conjugated todrugs. As described below, the drug of the ADC can be any number ofagents, including but not limited to cytotoxic agents such aschemotherapeutic agents, growth inhibitory agents, toxins (for example,an enzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), or a radioactive isotope (that is, aradioconjugate) are provided. In other embodiments, the inventionfurther provides methods of using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of a multispecific antibody and one or more small moleculetoxins, such as a maytansinoids, dolastatins, auristatins, atrichothecene, calicheamicin, and CC1065, and the derivatives of thesetoxins that have toxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in U.S. Pat. No. 5,416,064,WO/01/24763, 7,303,749, 7,601,354, U.S. Ser. No. 12/631,508,WO02/098883, 6,441,163, 7,368,565, WO02/16368 and WO04/1033272, all ofwhich are expressly incorporated by reference in their entirety.

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises a multispecific antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (see U.S. Pat. No. 6,884,869expressly incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF (see US 2005/0238649,5,767,237 and 6,124,431, expressly incorporated by reference in theirentirety).

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin γ1 as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see 4,169,888, incorporated by reference) and duocarmycins aremembers of a family of antitumor antibiotics utilized in ADCs. Theseantibiotics appear to work through sequence-selectively alkylating DNAat the N3 of adenine in the minor groove, which initiates a cascade ofevents that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302,and 7,507,420, all of which are expressly incorporated by reference.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include a multispecific antibodyas the Antibody unit, a drug, and optionally a linker that joins thedrug and the binding agent.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with anmultispecific antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e.g. amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker; in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: 509)). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment,” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the multispecificantibodies of the invention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachements (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of 3H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of 3H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the multispecificantibody of the invention can be evaluated in a suitable animal model.For example, xenogenic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al., 1997,Nature Medicine 3: 402-408). Efficacy can be measured using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecifcities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Thus for B cell tumors, the subject may experience a decrease in theso-called B symptoms, i.e., night sweats, fever, weight loss, and/orurticaria. For pre-malignant conditions, therapy with an multispecifictherapeutic agent may block and/or prolong the time before developmentof a related malignant condition, for example, development of multiplemyeloma in subjects suffering from monoclonal gammopathy of undeterminedsignificance (MGUS).

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the multispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an multispecific antibody used in the present invention is about0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as0.3, about 1, or about 3 mg/kg. In another embodiment, he antibody isadministered in a dose of 1 mg/kg or more, such as a dose of from 1 to20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the multispecific antibody is administered byinfusion in a weekly dosage of from 10 to 500 mg/kg such as of from 200to 400 mg/kg Such administration may be repeated, e.g., 1 to 8 times,such as 3 to 5 times. The administration may be performed by continuousinfusion over a period of from 2 to 24 hours, such as of from 2 to 12hours.

In one embodiment, the multispecific antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, ifrequired to reduce side effects including toxicity.

In one embodiment the multispecific antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the multispecific antibody.

In a further embodiment, the multispecific antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the multispecific antibody is administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In one embodiment, the multispecific antibody is administered by aregimen including one infusion of an multispecific antibody followed byan infusion of an multispecific antibody conjugated to a radioisotope.The regimen may be repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the multispecific antibody molecule thereof is usedin combination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,overexpressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

Examples

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

Example 1. Prototype “Triple F” Bispecific Antibody

The present invention describes novel immunoglobulin compositions thatco-engage a first and second antigen. One heavy chain of the antibodycontains a single chain Fv (“scFv”, as defined herein) and the otherheavy chain is a “regular” Fab format, comprising a variable heavy chainand a light chain (see FIG. 1). This structure is sometimes referred toherein as “triple F” format (scFv-Fab-Fc). The two chains are broughttogether by the dimeric Fc region (see FIG. 2). The Fc region can bemodified by amino acid substitution to allow for efficient purificationof the “triple F” heterodimer. Further, the Fc region can be modified byamino acid substitution to promote the formation of the “triple F”heterodimer. Examples of Fc substitutions are described more fullybelow.

Fc substitutions can be included in the “triple F” format to allow forefficient purification of the desired “triple F” heterodimer over theundesired dual scFv-Fc and mAb homodimers. An example of this is in theinclusion of Fc substitutions that alter the isoelectric point (pI) ofeach monomer so that such that each monomer has a different pI. In thiscase the desired “triple F” heterodimer will have a different pI thanthat of the undesired dual scFv-Fc and mAb homodimers, thus facilitatingisoelectric purification of the “triple F” heterodimer (e.g., anionicexchange columns, cationic exchange columns). These substitutions alsoaid in the determination and monitoring of any contaminating dualscFv-Fc and mAb homodimers post-purification (e.g., IEF gels, cIEF, andanalytical IEX columns). See FIG. 3 for a list of substitutions that canbe made in Fc monomer 1 and Fc monomer 2 to allow for efficientpurification of the desired “triple F” heterodimer.

Fc substitutions can be included in the “triple F” format to “skew” theformation toward the desired “triple F” heterodimer over the undesireddual scFv-Fc and mAb homodimers. For example, see FIG. 4 for a list ofsubstitutions that can be made in Fc monomer 1 and Fc monomer 2 to“skew” production toward the “triple F” heterodimer. Amino acidsubsitutions listed in FIG. 3 and FIG. 4 can be combined, leading to anincreased yield of “triple F” heterodimer that can be easily purifiedaway from any contaminating dual scFv-Fc and mAb homodimers.

After optimization of an scFv domain for inclusion in the “triple F”format, an optimized scFv domain can be coupled with a variety ofstandard antibody heavy chains in a convenient fashion. For example, ananti-CD3 scFv for recruiting T cell cytotoxicity can be coupled with avariety of anti-tumor antigen antibody heavy chains (e.g., those bindingCD5, CD20, CD30, CD40, CD33, CD38, EGFR, EpCAM, Her2, HM1.24, or othertumor antigen). Further examples of optimized scFv domains that can beconveniently coupled with standard antibody heavy chains includeanti-CD16 scFv for natural killer cell cytotoxicity; anti-CD32b scFv forinhibitory activity (here the coupled antibody heavy chain would bind,e.g., CD19, CD40, CD79a, CD79b, or other immune receptors); andanti-transferrin receptor scFv, anti-insulin receptor, or anti-LRP1 fortransport across the blood-brain barrier.

Example 2. Multi-Specific Antibodies Derived from the “Triple F” Format

Multi-specific antibodies can be constructed by attaching additionalscFv or Fab domains that bind a third antigen to the C-terminus of oneof the “triple F” heavy chains. See FIG. 5 for examples. Alternatively,the C-terminal scFv or Fab may bind the first or second antigen, thusconferring bivalency and an increase in overall binding affinity forthat antigen.

Multi-specific antibodies can also be constructed by coupling thescFv-Fc heavy chain of the “triple F” format may with rearrangedantibody heavy chains as depicted in FIG. 6. Such rearranged heavychains may include an additional Fv region that binds a third antigen oran additional Fv region that binds the first antigen or second antigen,thus conferring bivalency and an increase in overall binding affinityfor that antigen.

Example 3. Anti-CD19 Fab x Anti-CD3 scFv “Triple F” Bispecific

Amino acid sequences for anti-CD19 Fab x anti-CD3 scFv “triple F”bispecifics are listed in the figures. Amino acid substitutions made toallow for efficient purification of the desired “triple F” heterodimerover the undesired dual scFv-Fc and mAb homodimers are underlined. Aminoacid sequences for preferred humanized anti-CD3 variable regions arelisted in FIGS. 2 and 6 (with CDRs underlined). Some examples ofexpression and purification of the desired “triple F” species and itsbioactivity are given below.

The production of XENP11874, a “triple F” bispecific with an anti-CD19Fab and anti-CD3 scFv, is outlined in FIG. 9. In FIG. 9A, the ionexchange purification of the desired “triple F” heterodimer from theundesired dual scFv-Fc and mAb homodimers is shown. The purity of the“triple F” fraction was checked by IEF gel, (data shown in FIG. 9B ofU.S. Ser. No. 61/818,410, all figures and legends of which are expresslyincorporated by reference). Finally, SEC was used to confirm thehomogenous size of the “triple F” product (data shown in FIG. 9C of U.S.Ser. No. 61/818,410, expressly incorporated by reference).

XENP11874, anti-CD19 Fab x anti-CD3 scFv “triple F” bispecific, wasshown to have potent bioactivity. The ability of XENP11874 to potentlyrecruit T cells for B cell depletion is shown in FIG. 10 of U.S. Ser.No. 61/818,410, expressly incorporated by reference).

The production of XENP11924, a “triple F” bispecific with an anti-CD19Fab and anti-CD3 scFv, is outlined in FIG. 11 of U.S. Ser. No.61/818,410, expressly incorporated by reference. In FIG. 11A of U.S.Ser. No. 61/818,410, the ion exchange purification of the desired“triple F” heterodimer from the undesired dual scFv-Fc and mAbhomodimers is shown. The purity of the “triple F” fraction was checkedby IEF gel, shown in FIG. 11B (of U.S. Ser. No. 61/818,410). Finally,SEC was used to confirm the homogenous size of the “triple F” product(see FIG. 11C of U.S. Ser. No. 61/818,410).

XENP11924, anti-CD19 Fab x anti-CD3 scFv “triple F” bispecific, wasshown to have potent bioactivity. The ability of XENP11924 to potentlyrecruit T cells for the killing of the Raji tumor cell line is shown inFIG. 12 of U.S. Ser. No. 61/818,410.

Example 4. Anti-CD38 Fab x Anti-CD3 scFv “Triple F” Bispecific

Amino acid sequences for anti-CD38 Fab x anti-CD3 scFv “triple F”bispecifics are listed in FIG. 13 of U.S. Ser. No. 61/818,410. Aminoacid substitutions made to allow for efficient purification of thedesired “triple F” heterodimer over the undesired dual scFv-Fc and mAbhomodimers are underlined. Some examples of expression and purificationof the desired “triple F” species and its bioactivity are given below.

The production of XENP11925, a “triple F” bispecific with an anti-CD38Fab and anti-CD3 scFv, is outlined in FIG. 14 of U.S. Ser. No.61/818,410. In FIG. 14A of U.S. Ser. No. 61/818,410, the ion exchangepurification of the desired “triple F” heterodimer from the undesireddual scFv-Fc and mAb homodimers is shown. The purity of the “triple F”fraction was checked by IEF gel, shown in FIG. 14B of U.S. Ser. No.61/818,410. Finally, SEC was used to confirm the homogenous size of the“triple F” product (see FIG. 14C of U.S. Ser. No. 61/818,410).

XENP11925, anti-CD38 Fab x anti-CD3 scFv “triple F” bispecific, wasshown to have potent bioactivity. The ability of XENP11925 to potentlyrecruit T cells for the killing of the RPMI8226 tumor cell line is shownin FIG. 15 of U.S. Ser. No. 61/818,410.

Example 5. Identification and Repair of Destabilizing pI-AlteringIsotypic Constant Region Variants

As described above, efforts can be made to minimize the risk thatsubstitutions that increase or decrease pI will elicit immunogenicity byutilizing the isotypic differences between the IgG subclasses (IgG1,IgG2, IgG3, and IgG4). A new set of novel isotypes was designed based onthis principle. These new variants are called ISO(−), ISO(+), andISO(+RR). The thermal stability of these novel isotypes were determinedin a Hinge-CH2-CH3 (H-CH2-CH3) system (Fc region only). Proteins wereexpressed and purified as described above. Sequences for thisproof-of-concept system are listed in FIG. 16.

Thermal stability measurements (FIG. 17) determined by differentialscanning calorimetry (DSC) revealed that the ISO(−)/ISO(+RR) heterodimer(XENP12488, see FIG. 16 for sequence) was less stable than wild-typeIgG1 (XENP8156, see FIG. 16 for sequence). Subsequent engineeringefforts identified substitutions N384S/K392N/M397V in the ISO(−) heavychain as the source of the destabilization. As a result, the variantdesignated ISO(−NKV) was designed and tested (see FIG. 16). In thisvariant, positions 384, 392, and 397 were reverted to wild-type IgG1(S384N/N392K/M397V). The thermal stability of the ISO(−NKV)/ISO(+RR)heterodimer (XENP12757, see FIG. 16 for sequence) was measured by DSCand found to be equivalent to that of wild-type IgG1 (FIG. 17). Thisresult underscores the importance of choosing or not choosing particularpI-altering isotypic substitutions to avoid those that aredestabilizing.

Example 6. Additional Heterodimer-Skewing Fc Variants

As described above, heterodimer-skewing Fc variants can be made to biastoward the formation of the desired heterodimer versus the undesiredhomodimers. Additional heterodimer-skewing Fc variantsL368D/K370S-S364K/E357Q (XENP12760, see FIG. 18 for sequence) weredesigned and tested in a Hinge-CH2-CH3 system (Fc region only). Proteinwas expressed and purified as described above.

The proteins present after only a single standard protein A purificationstep were examined by high-performance liquid chromatography (HPLC)using a cation exchange (CIEX) column (see FIG. 19). This allowed thedetermination of the yield of desired heterodimer versus undesiredhomodimers. The presence of the L368D/K370S-S364K/E357Q variant(XENP12760, FIG. 19, bottom panel) introduced an extreme bias toward thedesired formation of heterodimer compared against the absence of thisvariant (XENP12757, FIG. 19, top panel). Note that heterodimer yield is95.8% with the L368D/K370S-S364K/E357Q variant versus only 52.7%without.

Additional heterodimer-skewing Fc variants were also designed andtested. FIG. 36 provides a list of engineered heterodimer-skewing Fcvariants with heterodimer yields (determined by HPLC-CIEX) and thermalstabilities (determined by DSC). The L368D/K370S-S364K/E357Q variantwith high heterodimer yield and high thermal stability is especiallypreferred.

Example 7. Additional Heterodimer-Skewing Fc Variants in the Fab-scFv-FcContext

Heterodimer-skewing Fc variants L368D/K370S-S364K/E357Q were engineeredinto an anti-CD19 x anti-CD3 Fab-scFv-Fc (see FIG. 15 for amino acidsequences). Control Fab-scFv-Fc XENP13228 lacked theseheterodimer-skewing Fc variants. The proteins present after only asingle standard protein A purification step were examined by anisoelectric focusing (IEF) gel. This allowed the determination of theyield of desired heterodimer versus undesired homodimers. The presenceof the L368D/K370S-S364K/E357Q variant (XENP13122, FIG. 22, right lane)introduced an extreme bias toward the desired formation of heterodimer(center band) compared against the absence of this variant (XENP13228,FIG. 22, left lane).

1.-81. (canceled)
 82. A heterodimeric antibody comprising: a) a firstmonomer comprising an anti-CD3 scFv-hinge-CH2-CH3 monomer, whereinCH2-CH3 is a first variant Fc domain and said anti-CD3 scFv comprises anscFv variable heavy chain comprising SEQ ID NO: 397, an scFv variablelight chain comprising SEQ ID NO: 398 and a charged linker comprisingSEQ ID NO: 450; b) a second monomer comprising a VH-CH1-hinge-CH2-CH3monomer, wherein VH is a first variable heavy chain and CH2-CH3 is asecond variant Fc domain; and c) a light chain comprising a firstvariable light chain; wherein said second variant Fc domain comprisesamino acid substitutions N208D/Q295E/N384D/Q418E/N421D, wherein saidfirst and second variant Fc domains each comprise amino acidsubstitutions E233P/L234V/L235A/G236del/S267K, and wherein said firstvariant Fc domain comprises amino acid substitutions S364K/E357Q andsecond variant Fc domain comprises amino acid substitutions L368D/K370S,wherein numbering is according to the EU index as in Kabat.
 83. Theheterodimeric antibody of claim 82, wherein said first and secondvariant Fc domains are variant human IgG1, IgG2 or IgG4 variant Fcdomains.
 84. The heterodimeric antibody of claim 82, wherein said firstand second variant Fc domains are variant human IgG1 Fc domains.
 85. Anucleic acid composition comprising: a) a first nucleic acid encodingsaid first monomer of claim 82; b) a second nucleic acid encoding saidsecond monomer of claim 82; and c) a third nucleic acid encoding saidlight chain of claim
 82. 86. An expression vector comprising said firstnucleic acid, said second nucleic acid and said third nucleic acid ofclaim
 85. 87. A host cell comprising said expression vector of claim 86.88. A method of producing the heterodimeric antibody of any one ofclaims 82 to 84 comprising culturing the host cell of claim 6 andrecovering the antibody from the cell culture.